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Storage proteins are present in the hemolymph from larvae and adults of the Colorado potato beetle.

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Archives of Insect Biochemistry and Physiology 20:119-I 33 (1992)
Storage Proteins Are Present in the Hemolymph
From larvae and Adults of the Colorado
Potato Beetle
Bertha Koopmanschap, Hans Lammers, and Stan de Kort
Department of Entomology, Agricultural University, Wqeningen, Netherlands
The protein composition of larval and adult hemolymph from the Colorado
potato beetle, Leptinotarsa decernlineata, was investigated and some abundant, high molecular weight proteins were identified and characterized. Diapause protein l , which occurs in the hemolymph of last instar larvae and
short-day adults, appeared to be a storage protein. This protein dissociated
into two bands due to the high pH used in nondenaturing gels. Its quaternary structure was established by chemical crosslinking. It appeared to be a
hexamer. Diapause protein 1 is composed of = 82,000 subunits. The amino
acid composition and N-terminal sequence of this protein has been determined. Specific antibodies against diapause protein 1 have been developed.
Topical application of 1 kg pyriproxyfen, a juvenile hormone analog, to last
instar larvae and short-day adults suppressed the appearance of this protein
in the hernolymph. Pyriproxyfen prematurely induced vitellogenin, when
applied to last instar larvae.
A larval specific protein was also identified i n the hemolymph. Its temporary appearance in the hemolymph of last instar larvae, its subunit composition (M, * 82,000) and its suppression by pyriproxyfen suggests that this protein
is a storage protein as well.
@1992WiIey-Liss, inc.
Key words: diapause proteins, hemolymph proteins, larval specific protein, pyriproxyfen
The protein compositionof the hernolymphfrom adult Leptinotmsa ~ e c e ~ ~ ~ ~ e u ~ f f
varies in beetles reared under different photoregimes. Under long-day condi-
Acknowledgments: We thank Wallace Clark (Biotechnology Center, University of Arizona, Tucson) for the amino acid analyses and N-terminal sequencing of the diapause proteins. The
critical remarks on the manuscript by Dr. john Law (University of Arizona, Tucson) and two
unknown referees are highly appreciated.
Received September 16,1991;accepted January31,1992.
Address reprint requests to C.A.D. de Kort, Department of Entomology, Agricultural University, P.O. Box 8031,6700 EH Wageningen, The Netherlands.
0 1992 Wiley-Liss, Inc.
Koopmanschap et al.
tions (18 h L*/6 h D), females start oviposition 5 days after adult emergence,
which is reflected by the appearance of vitellogenin in the hemolymph from
day 2 onwards. Under short-day conditions (10 h LA4 h D), which lead to
diapause 11-12 days after emergence, diapause proteins (short-day proteins)
are predominant in the hernolymph, whereas vitellogenin is hardly detectable
[l-31. Diapause proteins and vitellogenin are major proteins in the hemolymph
of this beetle. Another major protein in the hemolymph is lipophorin, the
primary lipid transport protein of insect hemolymph. Lipophorin from the
Colorado potato beetle was isolated and its molecular composition has been
characterized. It functions not only as a lipid transport vehicle, but in this
beetle it also transports JH [4].Thus the functions of vitellogenin and lipophorin
are relatively well known.
Less information exists about the function of diapause proteins. De Loof [2]
identified by disk electrophoresis three different diapause proteins in the
hemolymph of beetles reared under short-day conditions. One of these proteins (diapause protein 1)also occurs in last instar larvae. It disappears towards
the end of the pupal stage, suggesting that it is identical to so-called storage
proteins, described for other species [5-71. Dortland [3] arrived at the same
conclusion after he observed that diapause protein 1was stored in the fat body.
Insect storage proteins have been well studied in Diptera and Lepidoptera,
and different storage protein classes can be distinguished (for review see
[5]). Recently, Telfer and Kunkel [6] introduced the name hexamerin as a
descriptive and generic term that covers all hexamers of arthropods with
M, of approximately 500,000, the most prominent being the insect storage
hexamers and the hemocyanins. There is no information about the existence
of hexamerins in Coleoptera. Peferoen et al. [8] have reported the molecular
weights of the subunits of the most abundant proteins in the hemolymph
from the Colorado potato beetle, but their data do not reveal any value typical
for insect storage hexamers. Because data about the molecular weights of
the native proteins do not exist, we investigated these characteristics for a
number of abundant hemolymph proteins from the Colorado potato beetle.
Our studies revealed that diapause protein 1from the Colorado potato beetle
may be considered as an insect storage hexamer, which appearance is suppressed by JH.
A laboratory strain of the Colorado potato beetle, Leptinotarsa decemlineata
Say, was reared on fresh potato foliage at 25°C under long-day (18 h L/6 h D)
or at 23°C under short-day (10 h L/14 h D) conditions. Fourth instar larvae
*Abbreviations used: ApoLp I = apolipophorin I; apoLp I I = apolipophorin I I ; D = darkness; DSP = dithiobis(succinimidy1propionate);JH = juvenile hormone; JHA = juvenile hormone analog; L = light on; LSP = larval specific protein; PBS = phosphate buffered saline;
PMSF = phenylmethylsulfonyl fluoride; PTC-AA = phenylthiocarbamyl-amino acid; PTH =
phenylthiohydantoin; S D S = sodium dodecyl sulfate.
Hemolymph Storage Proteins
used in our experiments were taken from long-day cultures only. Under these
conditions, fourth instar larvae stop feeding 6 days after the last larval molt
and display digging behavior in preparation for pupation. Pupation takes place
in the soil and adults emerge 11-12 days after larval digging. Under long-day
conditions, newly emerged beetles immediately start feeding, mate between
day 3 and 4, and oviposit from day 5 onwards. Under short-day conditions,
adults also feed intensively, but do not show reproductive behavior. They stop
feeding and display digging behavior between days 11 and 12, indicating the
onset of diapause. Diapause lasts for at least 3 months.
Hemolymph was collected in capillary pipettes from precisely aged larvae
or adults after clipping a leg. The hemolymph samples were immediately diluted
to one-half in cold buffer containing 50 mM phosphate (pH 7.4),150 mM NaC1,
10 mM EDTA, 0.1 mM paraoxon, 0.1 mM PMSF, and a few grains of phenylthiourea. The protease inhibitor and paraoxon were kept in stock solutions
in ethanol and the solvent was evaporated immediately before dilution of the
hemolymph sample. Hemocytes were sedimented by centrifuging at 10,OOOg
for 4 min and the hemolymph was used immediately or stored at - 20°C.
The JHA, pyriproxyfen,2-[ 1-methyl-2-(4-phenoxyphenoxy)-ethoxy]pyridine,
was a 10% emulsifiable concentrate, supplied by Dr. H. Oouchi, Sumitomo
Chemical Co., Ltd., Osaka, Japan. Controls were treated with emulsifier solution, without JHA. Both solutions were stored at 4°C. For each application,
fresh solutions were prepared in acetone and applied to the insects in a volume of 1~ 1using
a microsyringe, operated with a repeating dispenser.
Gel Electrophoresis
Nondenaturing PAGE, using 7% continuous or 5.5-20% gradient slab gels,
was performed horizontally as described before IS], except that the buffer was
changed when indicated. SDS-PAGE was also performed horizontally with 6-20%
slab gels as described elsewhere [9] or vertically with the BIO-RAD Protean I1
slab cell (Richmond, CA), according to the instructions of the manufacturer.
The hemolymph samples for SDS-PAGE were further diluted with Laemmli
[lo] sample buffer containing 5% P-mercaptoethanol and heated to 100°C for
2 min, before application to the slots. The molecular weights of the proteins
were estimated by comparison with standard proteins of high and low molecular weight (Pharmacia, Uppsala, Sweden), using regression analysis.
Gel Permeation Chromatography
Chromatography was carried out at room temperature in a 2.6 x 40 cm column packed with Biogel A-1.5 m (BIO-RAD). The elution buffer was 50 mM
phosphate buffer (pH 7.2), containing 0.15 M NaCl and 0.02% sodium-azide.
Samples (ca. 30 mg of protein) were eluted at a flow of 30 mllh, controlled by
a LKB 2132 microperpex pump (LKB-Produkter AB, Bromma, Sweden) and
the absorbance of the eluant was monitored continuously at 280 nm using a
LKB 2138 Uvicord S.Fractions (2.5ml each) were collected with a LKB Ultrorac
fraction collector and the appropriate fractions were pooled and concentrated
by ultrafiltration, using an Amicon (Grace & Co., Amicon Division, Beverly,
MA) stirred cell system.
Koopmanschap et al.
Chemical Crosslinking
To study the quaternary structure of diapause protein 1, purified protein
(100 pg) was incubated for 1 h at room temperature in 100 pl of 0.2 M triethanolamine buffer (pH 7.5 or 8.3) containing 0.15 M NaCl and 0.5 or 0.05 mglml
DSP. After incubation 30 p1 of the reaction mixtures was diluted with twofold
concentrated SDS sample buffer without P-mercaptoethanol, boiled for 2 min,
and then applied to a 3-8% SDS gradient gel.
Antisera were prepared against LSP and diapause proteins 1A and lB, which
were designated as antisera 311, 291, and 292, respectively, The purified antigens were dissolved in 0.9% NaC1, emulsified with an equal volume of Freund’s
complete adjuvant, and injected subcutaneously into rabbits. After 5 weeks,
the same amount of antigen was emulsified in Freund’s incomplete adjuvant
and again injected. The same injection was repeated after another 3 weeks.
Eight days after the second booster injection, 40 ml of blood were collected
from each rabbit and serum was separated from the clot after one night in the
cold room. It took 5 booster injections to prepare a suitable antiserum
against LSP.
The protein fractions used for immunization were purified by PAGE and
eluted from the gels with a BIO-RAD Mode1 422 electro-eluter, using a volatile
ammonium bicarbonate buffer containing 0.1% SDS, according to the instructions of the manufacturer. Twenty slots, each containing 2 p1 of hemolymph,
were run in a nondenaturing 5.5-20% gradient slab gel as described above.
After staining of the gel for 30 min in 0.1% Coomassie R-250, dissolved in 10%
acetic acid, 40% methanol, and destaining of the gel in the same solvent, the
bands of interest were cut from the gel, sliced into small pieces, and eluted for
4-5 h with the electro-eluter. The eluted proteins were collected in 500 pl of
elution buffer per elution tube, transferred to microcentrifuge tubes, and freezedried. The dried protein fractions were dissolved in a small volume of SDS
sample buffer by heating at 100°C for 2 min and subsequently subjected to
SDS-PAGE, using a 6-20% gradient gel. After another round of staining and
destaining, the = 80,000 protein bands from each fraction were cut from the
gel, sliced, and eluted by the same procedure. After freeze-drying, the protein fractions were dissolved by boiling in a small amount of SDS sample
buffer and further diluted with 750 ~ 1 0 . 9 %
NaCl. This fraction was used for
injection after emulsification with Freund’s adjuvant. This protein fraction
contained a single band after Coomassie staining of Western blots from an
SDS gel.
Immunological tests were performed on whole hemolymph protein Samples or on purified protein samples after separation by SDS-PAGE and Western blotting of the proteins to Immobilon transfer membranes (Millipore, Bedford,
MA), as described before [4].After the transfer step, unoccupied binding sites
of the transfer membrane were blocked by incubating the membranes for 45
min at 37°C in a solution of 5% ( w h ) of skimmed milk powder in PBS (10 mM
phosphate (pH 7.4), 0.8% NaC1, 0.02% KC1, and 10 mM NaN3). After blocking, the membranes were washed 3 times in PBS containing 0.1% skimmed
milk for 5 min, and subsequently for 2 h at room temperature in the same
Hemolymph Storage Proteins
solution containing the primary antibody diluted by a factor of 2,000. After
this incubation, the blots were rinsed 3 times for 5 min in 0.1% skimmed milk
PBS solution. Detection of the immunologically responsive proteins occurred
with gold-labelled goat antirabbit immunoglobulin G (Janssen Life Sciences
Products, Beerse, Belgium), essentially as described before [4J.
Amino Acid Analysis and N-Terminal Sequencing
Samples were analyzed using an Applied BioSystems (Foster City, CA)Model
420A DerivatizerA30A Separation/92OAData Analyzer with automatic hydrolysis (Vapor Phase at 160°Cfor 1h 40 min) using pre-column PTC-AA analysis.
Samples were sequenced using an Applied BioSystems 477A ProteidPeptide
Sequencer (Edman chemistry) interfaced with a 120A HPLC (C-18 PTH, reversephase chromatography) Analyzer to determine PTH amino acids.
SDS Gel Electrophoresis
Figure 1 illustrates an SDS gel of hemolymph samples from different aged
larvae, short-day adults, and long-day males and females. The pattern shows
that most protein bands are similar in larvae and adults and in long-day and
short-day beetles. A number of these bands can be identified. ApoLp I and
apoLp I1 are indicated. Their M,s are 240,000 ? 10,000 and 77,000 ? 4,000 (n =
5), respectively, when calculated by comparison with the mobility of standard
proteins. ApoLp I and apoLp I1 are absent from larval samples, because they
were frozen before use. In freshly prepared larval hemolymph, these apoproteins
are normally present in SDS gels. Two vitellogenin subunits can be identified
with M,s of 199,000 k 5,000 and 162,000 h 5,000 (n = 5 ) , respectively. A prorninent band (indicated **) is visible adjacent to apoLp 11. It is present in fullgrown larvae and increases in concentration in short-day beetles, but is absent
in hemolymph from long-day beetles and day zero larvae. The M, of this protein band is = 82,000 ? 2,000 (n = 4). This, together with the appearance during certain developmental stages, suggests that this protein might be related
to insect storage hexamers. To investigate this, hernolymph samples from different stages were studied by nondenaturing gel electrophoresis.
Nondenaturing PAGE
Figure 2 illustrates the separation of hemolymph proteins from larvae and
short-day and long-day adults by a conventional 7% nondenaturing slab gel.
Three bands of relative high M, (M, 2 300,000) are present in the hemolymph
from larvae or short-day adults, which do not occur in long-day males or females.
These proteins are designated LSP (larval specific protein) and diapause protein 1A and lB, respectively. In addition, two other proteins are present in
the hemolymph of diapausing adults, whose M,s are relatively small on the
basis of their mobility in the gel. These are designated diapause protein 2 and
3, respectively. Their M,s are too small to conform to the definition of a hexamerin
and have not been studied further.
Figure 3 illustrates the protein pattern of hemolymph samples from longday males and females, last instar larvae, and short-day adults separated in a
Koopmanschap et al.
Fig. 1. SDS gradient gel of hemolymph samples from long-day females, day Iand day 6 lastinstar larvae (L4), and from different aged (2,6,10,90 days) short-day (SD) adults. S = standard
proteins of M, given in kDa. Apolp I and II = apolipophorin I and II; Vg = vitellogenin;
** = 82,000 subunit. The following amounts of hernolymph were applied: long-day female: 1 FI.
Larvae day 1 : 2 pi; day 6: 1 pi. Short-day beetles, day 2: 1 ~ lday
; 6 : 0.75 PI; day 10 and 90:0.5 pl.
Fig. 2. Conventional 7% slab gel of hemolymph samples from larvae (L4),diapausing adults
(D), and long-day males and females. S = high molecular weight standard proteins. Their M+
are given in kDa. LSP = larval specific protein; 1A and 1B = diapause protein 1A and 16;2 and
3 = diapause protein 2 and 3. In each slot 2 p1. of hemolymph was applied, except in D,where
1 pl was applied.
Hernolymph Storage Proteins
Fig. 3. Nondenaturing gradient gel (5.5-20%) of hernolymph samples from long-day males
and females, last-instar larvae (14) of day 1 or day 6, and different aged (2,6,10,90 days) shortday (SD) adults. S = standard proteins (see Fig. 2). Lp = lipophorin; LSP = larval specific protein;
1Aand 1B = diapause protein 1A and 16. The following amounts of hemolymph were applied:
long-day males and females: 1 PI. Day 1 larvae: 2 PIand day 6 larvae: 0.5 PI. Short-day beetles, day
2: 2 pl; day6: 1.5 pl; daylOand90: 1 PI.
nondenaturing 5.5-20% gradient gel at pH 8.9. Gradient gels offer a better
possibility to determine the M, of native proteins [ l l ] .
The M, values of the three high M, proteins were calculated by comparison
with standard proteins. LSP has a M, of = 614,000 5 29,000 (n = 6). The values for diapause protein 1A and 1B are = 465,000 4,000 (n 6) and 347,000
2 20,000 (n = 6), respectively. The M, of vitellogenin was also calculated and
was = 583,000 -I 18,000 (n = 7).
Diapause proteins 1A and 1B show unusual behavior in nondenaturing gels
at high pH. Comparison of Figures 2 and 3 indicates differences in staining
intensities, with diapause protein 1B more prominent in the gradient gel. Since
pH-dependent dissociation of insect storage hexamers is well known [7], we
ran another nondenaturing gradient gel at pH 8.3 instead of 8.9. Hemolymph
from different aged short-day beetles was used in this experiment. The results
are illustrated in Figure 4.At this pH, diapause protein 1A is much more prominent, which suggests that diapause protein 1B is a dissociation product of
diapause protein 1A rather than another protein.
To prove this more directly, the high molecular weight protein bands from
gradient gels (LSP, diapause protein 1A and 1B) were sliced after rapid staining and destaining of the gel and electroeluted. After electroelution and freezedrying, the protein samples were subjected to SDS gradient gel electrophoresis,
transferred to Immobilon, and stained by Coomassie. As shown in Figure 5A,
bopmanschap et al.
Fig. 4. Nondenaturing gradient gel (5.5-20%) of hemolymph from different aged short-day
beetles carried out at pH 8.3. The ages of the beetles and the amount of hemolymph applied
are similar to Figure 3. Abbreviations as in Figure 3.
the three protein bands all contain a single type of subunit of M, = 82,000.
Next, antibodies against the subunits of the three proteins were developed
and tested on Western blots from SDS gels, containing the following protein
samples: electroeluted bands from native gels representing LSP, diapause protein 1A and lB, whole hemolymph from last instar larvae, diapausing beetles, and long-day females. The results with antidiapause protein 1A serum
(serum 291) are illustrated in Figure 5B. It can be seen that it responds with
the subunits from diapause protein 1A and lB, but not with the subunit of
LSP. Also antidiapause protein 1 B serum (antiserum 292) crossreacted with
the subunits of 1A and lB, but not with LSP (not shown). Moreover, both
antisera responded with a single band of whole hemolymph from full-grown
larvae and short-day adults, but not with hemolymph from long-day females.
Anti-LSP serum reacted with the subunit of LSP, with a single band of whole
larval hemolymph, but not with hemolymph from day zero larvae or shortday adults (not shown). These studies suggest that diapause protein XA and
1B are composed of immunologicallyidentical subunits and that LSP is another
hemolymph protein. Further proof that diapause proteins 1A and 1B contain
the same subunit was derived from analysis of the amino acid composition
and N-terminal amino acid sequences of the two protein bands. The amino
acid compositions of diapause proteins 1A and 1B are very similar (Table l),
whereas the amino acid sequence of the first 20 amino acids of both proteins
Hernolyrnph Storage Proteins
Fig. 5. A: Western blots of SDS gradient gels of protein fractions electroeluted from native
gradient gels and stained with Coomassie R-250. 6: The same fractions plus whole hemolymph
from last-instar larvae (L4) and diapausing (D)
and long-day females were immunologically stained
with antidiapause protein 1A serum (291). The following amounts of protein fractions were
applied: A: LSP, equivalents of 0.8 pl of hernolymph; I A , equivalents of 2.0 pl; I B , equivalents of 1 .O kl. B: LSP, equivalents of 0.8 pI of hernolymph; I A , equivalents of 0.4 pl; I B , 0.2 PI;
L4,0.25 PI of hernolymph; D, 0.25 pl; females, 0.5 pl of hernolymph. Abbreviations as in Figure 3.
is exactly the same: Asn-pro-val-ala-asp-thr-asn-tyr-leu-lys-?-glu-gln-~n-ileleu-lys-leu-leu-tyr. The question mark on position 11 is probably cys or a
glycosylated amino acid.
From these experiments it can be concluded that diapause protein 1 B is a
dissociation product of diapause protein 1A, with the dissociation probably
due to the high pH used during electrophoresis of nondenaturing gels.
Structural Aspects of Diapause Protein 1
Since diapause protein 1 yielded two bands in nondenaturing gels, the question arises whether different forms of the protein occur under physiological
conditions. To study this, diapause protein 1was purified without using electrophoresis and subsequently chemically crosslinked. For this purpose hemolymph from diapausing beetles was centrifuged in a KBr gradient [4]. After
removal of lipophorin, the rest of the KBr gradient was diluted with phosphate buffered saline and concentrated by ultrafiltration. After another round
of dilution and concentration, to remove the bulk of KBr, the fraction was applied
to a gel permeation column. The first peak from this column appeared to be
pure diapause protein 1, which was verified by PAGE. After SDS-PAGE this
fraction yielded one band of M, = 82,000 (Fig. 6, lane 1). If this fraction is
incubated with high concentration of crosslinker (0.5 mg/ml), one band of high
Koopmanschapet al.
TABLE 1. Amino Acid Composition of Diapause Protein 1A and 1B From the Colorado
Potato Beetle
Amino acid
Aspartic acid
Glutamic acid
mol % protein 1A
mol % protein 1B
M, is visible in the SDS gradient gel (Fig. 6, lane 3). At 10 times lower crosslinker
concentration, six bands are apparent, which indicates that diapause protein
1 only occurs as a hexamer (Fig. 6, lane 2). Thus dissociation is probably an
artefact due to electrophoresis.
Effect of a JHA
Diapause protein 1occurs in the hemolymph of last instar larvae and shortday adults, two stages which are characterized by low JH titers [12]. To study
whether or not JH affects the appearance of diapause protein 1in the hemolymph, a JHA was applied topically to last instar larvae and short-day adults
and the effect on the protein pattern of the hemolymph was studied by
nondenaturing and SDS-PAGE. Nondenaturing gel electrophoresis was carried out at pH 8.3. Pyriproxyfen was used as JHA, because it had strong juvenilizing effects in larvae and adults of this beetle [13,14]. Larvae and young
adults were treated topically with 1 pg of pyriproxyfen on day 1 and day 3.
The protein pattern of the hemolymph was studied on day 6. The results with
larval hernolymph are illustrated in Figure 7. It can be seen from the native
gel (Fig. 7A) that the intensity of LSP and diapause protein 1decreased after
JHA treatment. In addition, a new band appeared with mobility very close to
LSP. This band is probably vitellogenin. Since LSP and vitellogen migrate very
close together in native gels, this region of the gel was sliced and electroeluted.
After electroelution, this fraction was subjected to SDS electrophoresistogether
with whole hemolymph from controls and pyriproxyfen treated larvae (Fig.
7B). The 82,000 subunit decreased in intensity after pyriproxyfen treatment
and two bands similar to the vitellogenin subunits are present. The lane with
the eluant from control larvae only contained the 82,000 subunit, whereas in
the lane with the sample from JHA treated larvae two additional bands are
visible with apparent M, similar to vitellogenin. That these subunits are indeed
derived from vitellogenin was shown by blotting and immunostaining using
HemolymphStorage Proteins
Fig. 6. SDS-PACE of diapause protein 1 after incubation with two concentrations of DSP. Diapause protein l (100 pg) was incubated with 0 (1),0.05(2)and 0.5 (3) mg/ml of crosslinker. After
1 h the reaction was stopped by addition of twofold concentrated SDS sample buffer without
p-mercaptoethanol and boiled for 2 min. An aliquot of each sample was electrophoresed at 30
mA in a 3-8% SDS gradient gel.
an antiserum developed against adult vitellogenin. The two vitellogenin subunits responded positively with the antiserum (not shown).
This paper describes the identification and characterization of some high
molecular weight proteins in the hemolymph of the Colorado potato beetle,
Leptinotarsa decemlineata .
Diapause protein 1, which has been reported previously by de Loof [2],
Dortland [3], and Peferoen et al. [ti],occurs as two separate bands on conventional nondenaturing slab gels and 5.5-20% gradient gels used in the current
study. With a high pH buffer system, diapause protein 1B stained more strongly
than diapause protein 1A (Figs. 2, 3). At lower pH, diapause protein 1A was
more prominent (Fig. 4). Subsequent experiments showed that the two bands
are probably derived from the same native protein.
Following electroelution, the two protein bands appeared to be composed
of subunits of similar M, (= 82,000), which also crossreacted immunologically
(Fig. 5A,B). Determination of the amino acid composition (Table 1) and the
Koopmanschap et al.
Fig. 7. Nondenaturing gradient gel with pH 8.3 electrophoresis buffer (A) and a 7% SDS gel
(6)of hernolymph samples (H) from control ( - 1 and pyriproxyfen ( ) treated larvae. Larvae
were treated on day 1 or day 3 and the hemolymph composition was analysed on day 6. All
slots contained 1 pI of hernolymph. Areas of the native gel containing LSP were electroeluted
from hernolymph samples of control (E -1 and pyriproxyfen (E +)treated larvae, lyophilized,
boiled in SDS sample buffer and subjected to electrophoresis. Lane L D 0 contains 1 PIof
hemolymph from long-day females. Apolp I and Apolp II = apolipophorin I and II, respectively; S = standard proteins (see Fig. 2); Vg = vitellogenin; ** = 82,000 subunit.
N-terminal sequence revealed that the two subunits are probably the same.
Calculation of the M, of the two protein bands suggests that diapause protein
1A is a hexamer (M, = 465,000) and 1B is a tetramer (M, 347,000). Dissociation, due to high pH, is well known for insect storage hexamers, particularly
in Diptera [7]. The combination of high pH (pH = 8.9 at room temperature)
and low ionic strength leads to dissociation of subunits, particularly during
long runs used for gradient gels (Fig. 3). At lower pH, dissociation of the hexamer
is much less (Fig. 4).The occurrence of the protein in different bands is probably an artefact due to nondenaturing electrophoresis. Direct proof that diapause protein l occurs only as a hexamer is derived from chemical crosslinking
experiments. Prior to chemical crosslinking, diapause protein 1was purified
in two steps without the use of electrophoresis. The purity of the protein was
checked by SDS-PAGE. The fraction appeared to contain one protein band after
SDS-PAGE (Fig. 5, lane 1).Subsequently, the purified fraction was subjected
to chemical crosslinking, using two concentrations of crosslinker. With high
concentration (0.5mg/ml), one band was visible after SDS gradient PAGE (Fig.
5, lane 3). If hexamers and tetramers are present together in the sample, chemical crosslinking would result in two bands with mobilities comparable to a
hexamer and a tetramer, respectively. With limiting concentrations of crosslinker
HemolymphStorage Proteins
(0.05mg/ml), six bands appeared after SDS-PAGE (Fig. 5, lane 2), which gradually decrease in intensity towards the higher oligomers. In addition, if the
oligomer numbers are plotted semilogarithmicallyagainst the distance of migration in the gel, a straight line is obtained, which strongly indicates that the
bands are different associations of the same monomer. Determination of the
M, of the different oligomers, by comparison with standard proteins, is not
possible, because interaction with the crosslinker affects the mobility of proteins during electrophoresis. Thus, this experiment demonstrates that diapause
protein 1, purified by a method without electrophoresis, occurs in a hexameric
form of = 82,000 subunits. The M, of the protein should be around 500,000.
Determination of the M, by native gel electrophoresis is therefore not accurate.
The hexameric configuration is direct proof that this protein belongs to the
group of hexamerins according to Telfer and Kunkel [6], and resembles the
insect storage hexamers in a number of biological and biochemical characteristics. It accumulates in the hemolymph during the last larval instar and its
concentration decreases during metamorphosis [2]. The protein also accumulates in the hemolymph of adults reared under diapause-inducing (short-day)
conditions (Figs. 1-3). During diapause it is present in the fat body as well
[15,161. The amino acid composition reveals a relatively high tyrosine/phenylalanine content (Table l), although the values are not as high as in storage
proteins from Diptera [6,7]. The fact that diapause protein 1 occurs in the
hemolymph of larvae and adults was shown previously by de Loof and
co-workers [2,8]. Recently, Wyatt [17] described the existence of a persistent
storage protein in larvae and adults of the migratory locust, Locusta migruforia.
Apparently, storage proteins may exist in adults, at least under certain physiological conditions.
The production of diapause protein 1is suppressed by a JHA, pyriproxyfen,
when applied to last instar larvae (Fig. 7) or short-day adults [14]. In a number of insect species, synthesis of storage hexamers is suppressed by JHA application to last instar larvae [18,19]. We recently described the suppression of a
larval storage protein by pyriproxyfen in last instar larvae of the migratory locust
[20]. Disappearance of the larval protein under the influence of JHA was accompanied by the precocious induction of vitellogenin in larvae. Our results confirmed an earlier report on vitellogenin induction in larvae of Locusfa migruforia
[21]. The experiments illustrated in Figure 7 demonstrate that pyriproxyfen
precociously induced vitellogenin when applied to last instar larvae of the Colorado potato beetle. This is the first report of vitellogenin induction in larvae
of holometabolous insects. The presence of vitellogenin in larval hemolymph
was demonstrated by nondenaturing and SDS-PAGEand by imrnunostaining.
Vitellogenin normally occurs in the hemolymph of long-day females after day
2. Its M,, determined by nondenaturing gels, is = 580,000, which is similar to
values found in other species [22]. After SDS-PAGE, two vitellogenin subunits
can be identified, with M,s of 199,000 and 162,000, respectively. The same
bands are present in larval hemolymph after application of 1pg pyriproxyfen,
but not in hemolymph from controls (Fig. 7). Both vitellogenin subunits
responded on Western blots with an antiserum developed against adult
A second storage protein, which is larval specific, is probably LSP. It is also
Kooprnanschap et al.
composed of -- 82,000 subunits, which were immunologically different from
the diapause protein subunits. A specific antiserum (serum 311) against this
protein has been developed. It responded with larval hemolymph, but not
with adult hemolymph. The apparent M, was determined by nondenaturing
gels to be = 614,000, which is too high for a hexamer with 82,000 subunits.
However, determination of the M, by native gel electrophoresis can be illusive, because the method measures the Stoke’s radius of the protein and can
yield misleading results if applied to proteins that are not spherical [ 6 ] .Moreover, the native protein may contain ligands, which affect the size of the molecule. One of us (H.L.) recently found that LSP contains 15% lipid. On this
basis, LSP is probably a hexamer, but further research on this point is required.
Lipophorin, the lipid transport and JH carrying protein of the hernolymph,
has been extensively discussed elsewhere [4].Its apoprotein composition has
been confirmed here. Larval lipophorin contains the same type of apoproteins
as adult lipophorin, but larval lipophorin is very sensitive to freezing and thawing, which damages the protein moiety of the protein. Its mass density (1.098
g/ml), determined by KBr gradient centrifugation [4], is similar to adult
lipophorin, and its intense red color suggests that it contains high concentration of carotenoids.
LSP has not been noticed by de Loof and co-workers [2,8], despite the fact
that de Loof [2] claimed that the band which he referred to as diapause protein 1is composed of several proteins. Our results differ from their data with
regard to the molecular weights of the subunits (apoproteins) of most of the
hemolymph proteins. They reported a molecular weight of 114,000 for the
subunit of diapause protein 1. Other data summarized in their Table 1 [8] are
also difficult to reconcile with our measurements. For example, they reported
that the most apparent chromoprotein (chromoprotein 2) is colored by orange
carotenoid pigments, constitutes 10-28% of the total protein concentration,
and occurs in eggs and hemolymph. We conclude from this that chromoprotein 2 is identical with lipophorin. They found three subunits (apoproteins)in
this fraction, yet the values of the M, of these apoproteins (356,000; 95,500;
90,000) do not agree with our data 141 nor with those from other lipophorins
[ 5 ] .We do not have an explanation for these discrepancies.
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adults, potato, beetle, protein, larvae, present, colorado, hemolymph, storage
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