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Isolation of the vitellogenin-binding protein from locust ovaries.

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Archives of Insect Biochemistry and Physiology 10:141-149 (1989)
Isolation of the Vitellogenin-Binding Protein
From Locust Ovaries
Axel Roehrkasten, Hans-Joerg Ferenz, Beate Buschmann-Gebhardt, and
JohannesHafer
Fachbereich Biologie, Universitaet Oldenburg, Federal Republic of Germany
A rapid, efficient procedure for the isolation and purification of the vitellogenin
binding protein from locust ovarian membranes is described. After solubilization with the nonionic detergent octyl-P-D-glucoside and removal of the
detergent, the binding protein is subjected to affinity chromatography on
vitellogenin coupled covalently to Affi-Gel 15. The binding protein is eluted
with suramin and EDTA at low pH value. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis reveals a polypeptide with a molecular weight
of 156,000 in the eluted fraction. By ligand blotting this polypeptide could be
identified as the vitellogenin binding protein. It retains its high-affinity binding properties. The specific binding of vitellogenin increases from 4.8 kg (intact
ovarian membranes) to 170.9 pg (affinity purified binding protein) per rng rnembrane protein, which corresponds to a purification factor of 35.
Key words: endocytosis, binding protein, vitellogenin, locust, affinity purification
INTRODUCTION
In egg-laying animals the predominant yolk protein precursors are called
vitellogenins. Insect vitellogenins are synthesized in the fat body released into
the hemolymph and transported to the ovaries 11-31. Maturing oocytes sequester vitellogenin by receptor-mediated endocytosis, a process first proposed by
Roth and Porter [4]for mosquito oocytes. Receptor-mediated endocytosis is a
common cellular process that includes the binding of a macromolecule, e.g.,
a protein, to a receptor molecule associated with the cell membrane and the
subsequent internalization of the receptor-ligand complex [5]. In several insect
species the selective uptake of vitellogenin by maturing oocytes could be
demonstrated [6-101 as well as the specific binding of vitellogenin to an oocyte
membranebound binding protein [ll-131, presumably the vitellogenin receptor.
Acknowledgments: The expert technical assistance of Mrs. Erika Tisler i s gratefully acknowledged. This study was supported by grants from the Deutsche Forschungsgemeinschaft to
H.-J. F.
Received March 17,1988; accepted December 9,1988.
Address reprint requests to Dr. Hans-JoergFprenz, Fachbereich Biologie, Universitaet Oldenburg,
Postfach 2503, D-2900 Oldenburg, Federal Republic of Germany.
0 1989 Alan R. Liss, inc.
142
Roehrkasten et a[.
Recently we were able to solubilize the locust vitellogenin-binding protein
from ovarian membranes by incubation with the nonionic detergent octyl-P-Dglucoside [14]. Studies with locust ovarian membranes and the solubilized binding protein molecule revealed that the binding protein is a trypsin and heatsensitive protein [12,141.
In the current study we describe a rapid, efficient procedure that yields a
vitellogenin-bindingprotein preparation that contains only minor impurities.
This purification procedure includes an affinity chromatography column containing vitellogenin covalently bound to Affi-Gel 15 and takes advantage of
the observation that binding protein-vitellogenin complexes can be dissociated with suramin [15].
MATERIALS AND METHODS
Animals
Locusta migratoria were obtained from ”Insektarium Dr. Frieshammer” (Jaderberg, F.R.G.). In the laboratory they were kept at 30°C with a daily photoperiod of 14 h and fed fresh grass or wheat shoots.
Solubilization of Membrane Proteins
Preparation and solubilization of locust ovarian membranes were as described
[9,12,14]. This membrane preparation consisted of oocyte membranes, follicular epithelia, basal membrane, and small amounts of oviduct membranes. In
a typical experiment, 103 mg membrane proteins obtained from 50 g locust
ovaries were suspended in 30 ml HEPES buffered saline (20 mM HEPES, 150
mM NaC1,l mM CaCI2, pH 7.4) containing 40 mM octyl-p-D-glucopyranoside
(Serva). After incubation for 10 min at 4°C the suspension was centrifuged at
100,OOOgfor 60 min. To the Supernatant, acetone (precooled to - 20°C)was added
with vigorous Vortex agitation (final acetone concentration 40%). The resulting precipitate was immediately collected by centrifugation at 20,OOOg for 20
min at 4°C. The pellets obtained were resuspended in HEPES buffered saline
by aspiration through a 22-gauge needle.
Affinity Chromatography
Locust vitellogenin was purified as described previously [9] and coupled
to Affi-Gel 15 (Bio-Rad) according to the specifications of the manufacturer.
About 13 mg of protein were covalently attached to 1 ml of gel. A column of
16 mm diameter containing 12.5 ml of the vitellogenin-Affi Gel was equilibrated with 200 ml of the HEPES buffered saline. Membrane extracts were
then applied to the affinity column and were recycled over the column for 4.5
h at room temperature. The column was then washed with 300 ml of HEPES
buffered saline at 4°C. Elution of the vitellogenin-binding protein was performed with 30 ml of a Tris-maleate buffer (50 mM, pH 6.0) containing 5 mM
suramin, 5 mM EDTA, and 150 mM NaCl at room temperature. After application of 4 ml of the buffer to the column, the flow was stopped for 15 min and
then the elution was continued and fractions (2 ml) were collected. Proteins
were precipitated with precooled acetone as described. The protein pellets of
Isolation of Vitellogenin-Binding Protein
143
fractions 4 to 10 were resuspended in HEPES-buffered saline, pooled, and
stored at - 70°C.
Polyacrylamide Gel Electrophoresis
Sodium dodecyl sulfate (SDS)* polyacrylamide gel electrophoresis was carried out with a LKB 2117 Multiphor according to LKB application note no. 306.
The slab gel contained 5% acrylamide and 0.1% SDS. The gel was stained with
silver nitrate as described by Wray et al. [16]. The gel was calibrated with the
following molecular weight standards (Sigma): myosin, 205,000; P-galactosidase, 116,000;phosphorylase b, 97,000; bovine serum albumin, 66,000; ovalbumin, 45,000; carbonic anhydrase, 29,000. The stained gel was scanned with
a computerized HeNe-laser densitometer (LKB) at 633 nm.
Ligand Blotting
After SDS electrophoresis of the affinity chromatography-purified binding
protein under nonreducing conditions, electrophoretictransfer of the protein
to nitrocellulose was carried out according to Towbin et al. [17] with a Bio-Rad
Transblot apparatus. Bovine serum albumin in Tris/HCI buffer (pH 7.4) was
used as the blocking agent. The blotting strips were incubated with locust
vitellogenin (1.2 mg/ml) for 60 min. Vitellogenin bound was visualized by the
PAP-method [MI,
including rabbit antiserum raised against vitellogenin as a
primary antibody (all other immunochemicalsexcept antivitellogeninwere purchased from Sigma).
Antibody Preparation
A polyclonal antibody directed against purified vitellogenin of Lucustu
rnigruturiu was prepared by immunizing a rabbit at intervals of 2 weeks with a
series of four injections of vitellogenin (3.5 mg protein for each injection). The
injections were given subcutaneously. Freund’s complete adjuvant was used
for the primary immunization; incomplete adjuvant, for the second and third
immunizations. The rabbit was bled 2 weeks after the third immunization by
heart puncture. IgG fractions were prepared from the serum by affinity chromatography on Protein-A-Sepharose [19].
Other Methods
Purified vitellogenin was radiolabeled with N-~uccinimidyl-[2,3-~H]-propionate (Amersham-Buchler)as described previously [9].Binding of [3H]propionylvitellogenin to soluble locust vitellogenin-bindingprotein was determined by
the filter assay method described [14]. Protein concentrations were determined
by the microassay procedure of the Bio-Rad protein assay.
RESULTS
Binding Activity After Acetone Precipitation of the Binding Protein
Before affinity chromatography, it was necessary to remove the detergent
because octyl glucoside disturbs the binding of the binding protein to the
*Abbreviationsused: LDL = low density lipoprotein; PAP = peroxidase-anti-peroxidase;SDS =
sodium dodecyl sulfate.
144
Roehrkasten et al.
vitellogenin coupled to the Affi-Gel beads of the affinity column. The detergent was removed by precipitating the binding protein with acetone. Figure 1
demonstrates that after addition of 40% acetone (final concentration) a maximum of about 85% of the solubilized membrane proteins can be recovered.
The acetone-precipitated proteins retained their full binding activity. Thus,
acetone precipitation does not denature the vitellogenin-bindingprotein and
can be used to remove contaminating chemicals and to concentrate the binding protein material.
Affinity Chromatography
Isolated ovarian membranes were solubilized in 40 mM octyl glucoside.
Unsolubilized material was removed by centrifugation. Membrane proteins
were precipitated by addition of ice-cold acetone to remove the detergent. The
precipitate was resuspended in 15 ml HEPES buffered saline and applied to a
column containing vitellogenin covalently coupled to Affi-Gell5. To elute the
binding protein from this affinity chromatography column, we took advantage of the observation that suramin, a polysulphonated aromatic compound
with a negative net charge, causes vitellogenin to dissociate rapidly from the
binding protein [15]. EDTA also prevents the binding of vitellogenin to the
binding protein [14]. Thus, the affinity column was eluted with a buffer containing 5 mM suramin and 5 mM EDTA. A strong major band is eluted from
the column consisting of one polypeptide (see below and Fig. 2, lane A). It
exhibited high-affinity binding for [3H]propionyl-vitellogeninwhen tested in
the filtration assay [14].
-
I
I
0
100
5
0
Y
U
c
ar
50
a
u
W
25 a
G
W
c
2
a
'loAcetone
Fig. 1. Effect of the acetone concentration on the specific precipitation of [3H]propionylvitellogenin binding activity. Aliquots of membrane proteins after solubilization and centrifugation (see Material and Methods) containing 661 pg of protein and 3.96 pg of specific
[3Hlpropionyl-vitellogenin
binding activity were diluted 1:4with modified HEPES-buffered saline
(final concentrations: 20 m M HEPES, 500 mM NaCI, 1 mM CaCI2, 10 mM octyl glucoside, pH
7.4). Precooled acetone (-20°C) was then added to obtain the indicated concentrations.
The precipitated pellets were resuspendedin 600 pI HEPES-buffered saline and assayed for specific [3Hlpropionyl-vitellogenin binding activity and protein content. The value for 100% of
total binding activity represents the amount of specific 13H] propionyl-vitellogenin binding activity that was extracted from unsolubilized oocyte membrane pellets after centrifugation.
Isolation of Vitellogenin-Binding Protein
A
116
97
B
145
C
b
66 *
45-
29-
Fig. 2. Silver stain of SDS-polyacrylamide gel electrophoresis of locust vitellogenin binding
protein at various stages of purification. Aliquots of oocyte membranes solubilized membrane
proteins and vitellogenin affinity purified fraction were denatured in the presence of 2-mercaptoethanol and separated on a 5% SDS polyacrylamide gel. The apparent molecular weight
of polypeptides was determined by the following standards (Sigma): myosin, 205,000; p-galactosidase, 116,000; phosporylase b, 97,000; bovine serum albumin, 66,000; ovalbumin, 45,000;
and carbonic anhydrase, 29,000. Lane A: eluate from vitellogenin Affi Gel-I5 (1.6 pg); lane B:
supernatant after octyl glucoside solubilization of oocyte membranes (5.4 pg); lane C: intact
oocyte membranes (24 pg).
Identification and Characterization of the Vitellogenin-Binding Protein
Figure 2 compares the SDS-electrophoretic mobility of the ovarian membrane protein extract at various stages of purification. The complex membrane
protein pattern of intact ovarian membranes (solubilized in SDS) is illustrated
in lane C of Figure 2. Nearly all membrane proteins are still present after solubilization with octyl glucoside (lane B). Lane A shows the gel profile of the
vitellogenin-binding protein eluted from the vitellogenin affinity column. The
eluted vitellogenin-binding protein is a single polypeptide with an apparent
molecular weight of 156,000. Only minor impurities contaminating the purified binding protein preparation can be detected with the sensitive silver stain
followed by densitometer tracing of lane A (Fig. 3).
Ligand blotting was used to visualize the vitellogenin-binding protein after
one-dimensional electrophoresis. By blotting the affinity-purifiedvitellogeninbinding protein (see Fig. 2, lane A), followed by treatment with vitellogenin
and by visualization of bound vitellogenin with the PAP-method, we obtained
146
Roehrkasten et al.
c
L
d
QI
U
t
b
n
L
0
v,
t
c
n
a
i
0
L
Fig. 3. Densitometric recording of lane A in Figure 2 (eluate from vitellogenin Affi-Gel-15) of
the silver-stained SDS-polyacrylamidegel.
one stained band identical to the 156,000band found after SDS-electrophoresis
(Fig. 4A). As a control, the ligand blotting was performed with the omission
of incubation with vitellogenin, which confirmed the specificity of the ligand
blotting method (Fig. 4B).
Estimates of Total Yield and Fold-Purificationfor the Purified Binding Protein
Table 1 summarizes the results of purification by the described procedure.
The intact ovarian membranes bound 4.8 pg of [3H]propionyl-vitellogeninper
mg of membrane protein. Half the initial membrane protein was recovered in
the octyl glucoside solubilized extract. After acetone precipitation, a slight
increase in binding activity was found (6.2 p,g [3H]propionyl-vitellogeninper
mg membrane protein), but total high-affinity binding decreased by 38%,
compared with the crude membranes. When the membrane proteins reconstituted in HEPES buffer were passed through the vitellogenin-Affi Gel 15
column, a total of 290 pg of protein was recovered (0.28% of the initial material). The affinity-purified binding protein had a specific binding activity of
170.9 pg [3H]propionyl-vitellogeninbound per mg of binding protein. This
represents a 35-fold purification, with an overall yield of 10%. However, because
of a possible incomplete retention of the binding protein molecule on the filters, the calculations can only be approximate.
DISCUSSION
During vitellogenesis, maturing insect oocytes take up selected hemolymph
proteins. The specific incorporationis accomplished by a process called receptor-
Isolation of Vitellogenin-Binding Protein
147
B
A
11697 *
Fig. 4. Identification of the vitellogenin binding membrane protein by ligand blotting. After
SDS-electrophoresis, proteins were transferred to nitrocellulose. Ligand blotting and visualization by the PAP method were performed as described. Lane A: incubation with vitellogenin;
lane B: incubation without vitellogenin. The positions of molecular weight standards are
indicated.
mediated endocytosis. Previous observations that isolated intact locust ovarioles
bind and incorporate vitellogenin [9] could be confirmed by studies with cellfree systems. Locust oocyte membranes possess specific and saturable binding sites for vitellogenin [12]. Similar observations have been made for the
TABLE 1. Purification of the Vitellogenin-Binding Protein From Locust Oocytes
Fraction
Intact
membranes
Solubilized
membranes‘
Vitellogenin
affinitycolumn‘
Loaded
(mg)
Protein
Recovered
(mg)
(%)
Binding activity
Specific”
Totalb
(ug/mg) (ug/mg)
(%)
-
103
100
4.8
498
100
102
49
48
6.2
307
62
170.9
50
10
48
0.29
0.28
Purification
factor
(-fold)
1
1.3
35
‘The values for specific binding represent pg [3H]propionyl-vitellogeninbound per mg protein
after incubation for 80 min at 24°C and after subtraction of the value for unspecific binding.
bTotal binding activity was calculated by multiplying specific binding activity by total protein
content.
‘Amount after protein was subjected to acetone precipitation.
148
Roehrkasten et al.
cockroach Nauphoeta cinerea [ll]and the tobacco hornworm Manduca sexta [13].
The locust vitellogenin binding protein could be identified as a membrane protein that could bind vitellogenin with high affinity even after solubilization of
the membranes with the detergent octyl glucoside [14].
Locust ovarian membranes possess a complex polypeptide pattern. However,
in a one-step procedure the octyl glucoside-solubilized vitellogenin-binding
protein could be considerably purified. By passing the solubilized membrane
proteins over a vitellogenin affinity chromatography column, one predominant protein could be eluted. SDS-polyacrylamideelectrophoresis of the eluted
protein followed by the very sensitive silver staining confirmed the presence
of a 156,000 protein, presumably the vitellogenin-binding protein.
In its nearly purified form the vitellogenin-binding protein retained the properties that are characteristic of the vitellogenin-binding protein in intact ovarioles [9], in oocyte membrane preparations [12], and in crude solubilized extracts [14]. The isolated binding protein continued to show a high affinity for
vitellogenin.
The recovery rate and the purification factor (35x ) of the binding protein is
not very high. This may be attributed in part to the fact that we used locust
ovarioles for the preparation of the vitellogenin-binding protein containing
oocytes, follicle cells, and the basal lamina. The follicle cells probably contribute a significant fraction of the initial total membrane proteins. Furthermore,
we assume that some of the binding protein material is incompletely retained
and lost during the filter assay, which also results in an underestimation of
the amount of high-affinity binding. In addition, after removal of the detergent the binding protein possibly aggregates to form micelles that may not be
bound well enough during the affinity chromatography, causing loss of material. Such micelles may also mask the binding sites of some binding protein
molecules, thereby reducing considerably the amount of vitellogenin that can
specifically be bound. Thus, at present it is difficult to correct for this low recovery or binding activity. More studies are needed to clarify these issues. Now
we shall try to improve the affinity chromatography methods to obtain more
and better purified vitellogenin binding protein. A more comprehensive characterization of that binding protein will be possible then.
Several properties of the locust vitellogenin-binding protein are similar to
those of the LDL-receptor system [20]. We have experimental evidence that
the vitellogenin-binding protein also carries negative residues that interact specifically with vitellogenin: trypan blue and suramin compete for binding to
vitellogenin [151; chemically modified vitellogenin (positive charges removed)
cannot be bound [14], (unreported data). The events following internalization
are not well known. Usually the schematic view reported for the LDL-receptor
is adopted [21]. Having considerably concentrated and punfied the vitellogeninbinding protein, we shall be able to investigate the fate of this binding protein
after endocytosis. Although many more studies will be necessary to understand fully the mechanism of binding protein function and the dynamics of
binding protein internalization and recycling, insect oocytes appear to be very
valuable for studying and understanding receptor-mediated endocytosis in any
cell type.
Isolation of Vitellogenin-BindingProtein
149
LITERATURE CITED
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Insect Physioll4,49 (1979).
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and Pharmacology. Gilbert LD, Kerkut GA, eds. Pergamon Press, New York vol 1, pp 83-111
(1985).
4. Roth TF, Porter KR: Yolk protein uptake in the oocyte of the mosquito Aedes uegypfi. J Cell
Biol20,313 (1964).
5. Goldstein HJ, Anderson RGW, Brown MS: Coated pits, coated vesicles, and receptor-mediated
endocytosis. Nature 279,679 (1979).
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insects. J Insect Physiol22,809 (1976).
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oocytes of Locusfu rnzgraforiu. J Insect PhysiolZ7, 869 (1981).
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locust oocytes. Wilhelm Roux’s Arch 195,411 (1985).
10. Kulakosky PC, Telfer WH: Selective endocytosis, in vitro, by ovarian follicles from Hyulophoru
cecropia. Insect Biochem 17, 845 (1987).
11. Koenig R, Lanzrein B: Binding of vitellogenin to specific receptors in oocyte membrane preparations of the ovoviviparous cockroach Nuuphoefa cinereu. Insect Biochem 15,735 (1985).
12. Roehrkasten A, Ferenz H-J: Properties of the vitellogenin receptor of isolated locust oocyte
membranes. Int J Inv Reprod Dev 10,133 (1986a).
13. Osir EO, Law JH: Studies on binding and uptake of vitellogenin by follicles of the tobacco
homworm, Munducu sexfu. Arch Insect Biochem Physiol3,513 (1986).
14. Roehrkasten A, Ferenz H-J: Solubiliation of the locust vitellogenin receptor. Biochem Biophys
Acta 860,577 (1986b).
15. Roehrkasten A, Ferenz H-J: Inhibition of yolk formation in locust oocytes by trypan blue
and suramin. Wilhelm Roux’s Arch 196, 165 (1987).
16. Wray W, Boulikas T, Wray VP, Hancock R Silver staining of proteins in polyacrylamide gels.
Anal Biochem 118,197 (1981).
17. Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide
gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci 76 (9),
4350 (1979).
18. Sternberger LA: Immunocytochemistry. Wiley, New York, Chichester, Brisbane, Toronto, p
354 (1979).
19. Tijssen P: Practice and theory of enzyme immunoessays. In: Laboratory Techniques in Biochemistry and Molecular Biology. Burdon RH, Knippenberg PH, eds. Elsevier, Amsterdam,
New York, Oxford 15, p 105 (1985).
20. Schneider WJ, Beisiegel U, Goldstein JL, Brown MS: Purification of the low density lipoprotein receptor, an acidic glycoprotein of 164,000 molecular weight. J Biol Chem 257,2664 (1982).
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232,34 (1987).
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