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Characterization of vitellin, egg-specific protein and 30 kDa protein from Bombyx eggs, and their fates during oogenesis and embryogenesis
Biochimica et Biophysica Acta 882 (1986) 427-436
427
Elsevier BBA 22354
Characterization of vitellin, egg-specific protein and 30 kDa protein from Bombyx eggs, and their fates during oogenesis and embryogenesis Jiang Zhu *, Leslie S. Indrasith and Okitsugu Yamashita ** Laboratory of Sericultural Science, Faculty of Agriculture, Nagoya University, Chikusa, Nagoya 464 (Japan) (Received January 20th, 1986)
Key words: Vitellin; Egg-specific protein; 30 kDa protein; Antigenicity; Oogenesis; Embryogenesis; (B. mori egg)
Three major yolk proteins, vitellin, egg-specific protein and 30 kDa proteins, were purified from the same extracts of Bombyx mori eggs by high-performance liquid chromatography on a molecular sieving column. Each preparation was judged to be homogeneous by polyacrylamide gel electrophoresis. The subunit structure was estimated to be as follows: vitellin is a tetramer with a molecular mass of 420 kDa, consisting of two heavy subunits (178 kDa) and two light subunits (43 kDa); egg-specific protein is a trimer (225 kDa) of two heavy subunits (72 kDa) and one light subunit (64 kDa); 30 kDa proteins are a mixture of three monomers (I, 2 and 3) consisting of respective subunit molecular masses of 32.0, 31.0 and 29.5 kDa. The three yolk proteins contained the usual amino acids together with various lipids and carbohydrates. Antisera to each protein did not cross-react. The titration of vitellin, egg-specific protein and 30 kDa proteins on rocket immunoelectrophoresis showed a differential accumulation pattern during the course of oogenesis. In newly laid eggs, vitellin, egg-specific protein and 30 kDa proteins accounted for approx. 40%, 25% and 35%, respectively, in weight. The eggs developed in male hosts after implantation of ovary discs were deficient in vitellin but contained egg-specific protein and 30 kDa proteins at comparable levels to the normal female eggs. During embryogenesis, egg-specific protein was rapidly and completely utilized. Approx. 35% vitellin and 50% 30 kDa proteins remained unused and were carried over to the hatched larvae. Such accumulation and utilization of yolk proteins are correlated with the fates of the proteins during oogenesis and embryogenesis of B. mori.
Introduction
The eggs of oviparous animals are generally characterized by a high accumulation of a single species of the protein vitellin [1,2]. In many insect species, vitellin accounts for more than 80% of the total yolk proteins, and is considered to be the sole storage reserve for subsequent embryogenesis [1,2,4]. In eggs of some lepidopteran insects, in* Present address: Suzhou Sericultural College, Suzhou, China. ** To whom correspondence should be addressed. Abbreviation: PMSF, phenylmethylsulfonyl fluoride.
cluding silkworms, vitellin comprises only about half of the total yolk proteins; the yolk contains significant amounts of other kinds of proteins [5-81. There are many reports on the physicochemical properties of insect vitellins, and they are now classified into three groups depending on the molecular structure [9]. Silkworm vitellin was purified from mature eggs and characterized to be a vitellin belonging to group 1 which consists of two heavy subunits and two light subunits [10]. Recently, Takesue et al. [11] reported a higher molecular weight for silkworm vitellin, but did not give any information on its subunit structure. The
0304-4165/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
428 second major yolk protein group of silkworm eggs consists of the non-sex-linked serum proteins with molecular masses of approx. 30 kDa [12] (referred to as 30 kDa proteins). These proteins are produced in the fat body and released into the hemolymph towards larval maturation and are finally sequestered into developing oocytes [5,10,12]. Izumi et al. [12] purified 30 kDa proteins from the hemolymph of mature silkworm larvae and characterized their molecular properties. However, no attempt was made to purify and characterize them from eggs, although their behavior on polyacrylamide gel electrophoresis seemed to be quite similar. The third main protein of silkworm eggs is the so-called egg-specific protein, which is produced by the ovary itself and accumulates especially in developing oocytes [6,13]. This protein was purified from eggs, and its structural characteristics were examined by Irie and Yamashita [6]. The more important physiological events during oogenesis and embryogenesis are dependent on this yolk protein [6,14]. To consider further their physiological roles, it is desirable to identify each protein in the same egg sample. In the present study, we purified sequentially 30 kDa proteins, vitellin and eggspecific protein from the same crude extracts and characterized their molecular properties. Further, using specific antisera, the developmental changes in the protein titres were specified on the same materials throughout oogenesis and embryogenesis. Materials and Methods
Preparation of hemolymph, ovaries and eggs. Three hybrid races (Shunrei x Shogetsu, Kinshu x Showa and Nichi 106 x Daizo) of the silkworm, Bombyx mori, were used and their embryonic development was progressed at a high temperature (25°C) to obtain diapause eggs at the next generation. The larvae were reared on fresh mulberry leaves or an artificial diet (Takeda Chemical Co., Tokyo) at 25 o C. The larval life took 27 days and adults emerged 10 days after pupal ecdysis. In some experiments, ovarian discs were transplanted into male 5th instar larvae to obtain vitellin-deficient eggs [14]. Developing ovaries were prepared daily throughout pupal-to-adult development [15].
Hemolymph was collected from male and female pharate adults of day 5, when the active vitellogenesis occurred in females. The female moths were allowed to lay eggs for a period of 3 h, and the newly laid eggs were stored at - 7 0 ° C for purification of yolk proteins or incubated at 25 °C to allow embryogenesis to occur. HC1 treatment was performed on day 1 to avoid diapause [16]. In these eggs larval hatching occurred mainly on day 10. Preparation of crude extracts. All procedures were carried out at 4°C or in ice, unless specified. Developing ovaries and eggs were homogenized in 5 vol. buffer A (50 mM phosphate buffer (pH 7.2)/0.4 M KC1/1 mM PMSF) using first a mortar and then a glass-Teflon homogenizer. The homogenate was centrifuged at 16000 x g for 20 min and the supernatant was used as the crude extract for purification and titration of yolk proteins. Hemolymph was diluted 5-fold with buffer A and processed as the ovary homogenate. DEAE-cellulose column chromatography. The crude extracts were prepared from about 20 g of eggs and dialyzed against three changes of buffer B (buffer A without KC1) overnight. The dialyzed solution was applied onto a column (2.5 (internal diameter) x 65 cm) of DEAE-cellulose (DE-23, Whatman Inc., Clifton, N J) which was preconditioned with buffer B. The column was washed with buffer B and eluted with 0.2 M KC1 and then 0.5 M KC1. Flow rate was 40 m l / h and a 10 ml fraction was collected. Proteins were monitored by absorbance at 280 nm and mobility on polyacrylamide gel electrophoresis. Each fraction was pooled and concentrated by a 60% saturation of ammonium sulfate. The fractions washed out from the first DEAE-cellulose column were dialyzed against three changes of buffer C (10 mM phosphate buffer (pH 8.0)/1 mM PMSF) and applied onto the second DEAE-cellulose column (1.3 (i.d.) x 25 cm) pre-equilibrated with buffer C. The column was developed with a linear gradient of 0-0.1 M KC1 at a flow rate of 40 m l / h . Protein fractions were pooled and concentrated by ammonium sulfate as above. High-performance liquid chromatography. Each protein sample from DEAE-cellulose column chromatographies was subjected to HPLC (Tri-
429 rotar III, Japan Spectroscopic Co., Tokyo) equipped with a molecular sieving column (7.5 mm (i.d.)x 60 cm; TSK gel-G 3000 SW, Toyo Soda Co., Tokyo). A 50/~1 sample (approx. 1.5 mg proteins) was injected at once and was eluted with buffer D (buffer B containing 0.1 M sodium sulfate) at a flow rate of 1 m l / m i n under a constant pressure of 30 k g / c m 2. Each peak was collected and concentrated using a centriflow filter (Amicon Co., Danvers, MA).
Sephadex G-200 and Sephacryl S-300 column chromatography. The purified yolk proteins were applied to a Sephadex G-200 column (1.6 (i.d.) x 100 cm) and a Sephacryl S-300 column (1.6 (i.d.) x 90 cm). The respective columns were eluted at a flow rate of 8 m l / h with 50 mM phospahte buffer (pH 7.5) containing 50 mM NaC1 and buffer B. Distribution coefficients (Kay) of proteins were determined from the void volume (V0) estimated by Blue dextran and the column volume (Vt) by tyrosine. The native molecular weights were calibrated by plotting Kav vs. log molecular weights of the standard proteins (Pharmacia Fine Chemicals, Uppsala). Polyacrylamide gel electrophoresis. Non-denatured electrophoresis of purified vitellin, eggspecific protein, 30 kDa proteins and crude extracts was carried out on a 6% polyacrylamide slab at pH 9.5 [17]. SDS-polyacrylamide gel electrophoresis was performed on 8.5%, 10% and 11.5% polyacrylamide gels containing 0.1% SDS [18]. Proteins were stained with Coomassie brilliant blue R-250 (Sigma Chemicals Co., St. Louis, MO). Relative staining intensity of bands was determined by a computing densitometer (Gelman Instrument Co., Ann Arbor, MI). Molecular weights were estimated using high- and lowmolecular-weight calibration kits (Pharmacia Fine Chemicals, Uppsala). Immunochemical analysis. Rabbits were injected subcutaneously at multiple sites with 5 m g / 5 ml of each purified protein mixed with an equal volume of Freund's complete adjuvant (Difco Laboratories, Detroit, MI), and after 30 days a booster injection was given. One week later the serum was obtained and stored at - 7 0 ° C . The anti-125 kDa egg-specific protein serum provided by Irie and Yamashita [6] was used in some experiments.
Immunodiffusion was carried out in 1.2% agar for 2 days according to the method of Ouchterlony and Nilson [19]. Rocket immunoelectrophoresis was run for 24 h at 7 mA using 1.0% agarose in 0.5 M barbital buffer (pH 8.5) containing 0.8% antiserum [20].
Determination of amino acids, lipids, carbohydrates and proteins. A lyophilized protein sample was hydrolyzed in vacuo in 6 M HCI at 165°C for 30 min. The sample was evaporated to dryness and dissolved in 0.2 M citrate buffer (pH 2.2). This solution was subjected to an amino acid analyser (JLC-68A, JEOL, Tokyo). Total lipids were extracted three times from the purified proteins (35-40 mg) with 3 ml mixture of chloroform and methanol (2 : 1, v/v) [21]. After washing with distilled water, the remaining residues were measured gravimetrically. Total carbohydrates were estimated using the anthrone method [22], and amounts were expressed as mannose equivalent. Protein content was determined by the method of Lowry et al. [23] using bovine serum albumin as a standard. Results
Identification of yolk proteins Fig. 1A shows a native polyacrylamide gel electrophoresis analysis of yolk protein extracts from the newly laid eggs along with serum proteins from male and female pharate adults. Female serum proteins (lane b) were separated into four main bands, numbered 1 to 4 in order of moving distance, but band 1 was completely deficient in male serum (lane a). Egg extracts (lane c) showed one additional main band (band 5). These tissueand sex-dependent banding patterns suggest that band 1 is from vitellogenin/Vtn [10], band 5 from egg-specific protein [6] and bands 2, 3 and 4 from 30 kDa proteins [12]. The SDS-polyacrylamide gel electrophoresis (Fig. 1B) of yolk proteins (lane c) gave seven main bands, numbered 1 to 7, and some minor bands. Bands 1 and 4 had respective molecular masses of 178 and 43 kDa (cf. Table I), and were completely deficient in male serum (lane a), thus corresponding to heavy and light subunits of vitellin [10]. Bands 2 and 3, with respective molecular masses of 72 and 64 kDa, were estimated to be
430
A
!n
B
jj
I
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I
e
6 o
Q
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Fig. 1. Banding patterns of proteins on native polyacrylamide gel electrophoresis (A) and an SDS-polyacrylamide gel electrophoresis (B). Protein samples (50-60 ktg) were from hemolymph of 5-day-old male pharate adults (a), hemolymph of 5-day-old vitellogenic females (b), newly laid eggs (c), purified vitellin (d), purified egg-specific protein (e), purified 30 kDa protein-1 (f), -2 (g), -3 (h). Native polyacrylamide gel electrophoresis (A) was done using 6% polyacrylamide gels and the major bands 1-5 in the migration order corresponded to vitellin, 30 kDa protein-l, 30 kDa protein-2, 30 kDa protein-3 and egg-specific protein, respectively. The polypeptide bands on SDS-polyacrylamide gel electrophoresis using 11% gels (B) were estimated as follows: band 1, heavy subunit (178 kDa) of vitellin; band 2, heavy subunit (72 kDa) of egg-specific protein; band 3, light subunit (64 kDa) of egg-specific protein; band 4, light subunit (43 kDa) of vitellin; bands 5, 6 and 7, polypeptide of 30 kDa protein-1 (32.0 kDa), -2 (31.0 kDa) and -3 (29.5 kDa), respectively.
subunits of egg-specific protein, because of their absence in serum samples (lanes a and b) [6]. Bands 5, 6 and 7, migrating with molecular masses of appro. 30 kDa, were identified as 30 k D a protein-l, -2 and -3 [12]. Consequently, the yolk of silkworm eggs consisted of the three groups of proteins, vitellin, egg-specific protein and 30 k D a proteins, which differed with regard to tissue distribution, sex-linkage and molecular weights.
Purification of yolk proteins The first DEAE-cellulose column chromatography of the egg extracts (Fig. 2) showed that not all c o m p o n e n t s of the 30 k D a proteins b o u n d to the column. Vitellin was eluted at 0.2 M KC1 and egg-specific protein at 0.5 M KC1. The separated proteins were precipitated with 60% a m m o n i u m sulfate and dissolved in the following buffer solutions: 30 k D a proteins in buffer C; vitellin in
buffer A; and egg-specific protein in buffer B. The fractions of vitellin and egg-specific protein were directly subjected to H P L C as described below. The 30 k D a protein fractions were again subjected to the second DEAE-cellulose column chrom a t o g r a p h y which was developed by a linear gradient (0-0.1 M KCI in buffer C) (Fig. 3). 30 k D a protein-1 was recovered by the buffer washing, 30 k D a protein-2 was eluted at 0.05 M KC1, and 30 k D a protein-3 at 0.08 M KC1. Each fraction was concentrated and used for the final purification using H P L C . The prepurified vitellin, egg-specific protein and 30 k D a proteins were subjected to H P L C which was developed with buffer D at a flow rate of 1 m l / m i n and were monitored at 280 nm (Fig. 4). Each protein was eluted as a single peak at the following specific retention times: 13 min for vitellin, 16 min for egg-specific protein, 21 min for 30
431 141
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160 80 120 Fraction number Fig. 2. Separation of 30 kDa proteins, vitellin (Vtn) and egg-specific protein (ESP) from the newly laid egg homogenates by the first DEAE-cellulose column chromatography. The dialyzed crude extracts from about 20 g of eggs were applied to the column (2.5 (i.d.)× 65 cm) pre-equilibrated with buffer B and washed with the same buffer. Further elution was done stepwise using the same buffer containing 0.2 M KC1 and then 0.5 M KCI. The column was developed at a flow rate of 40 m l / h with a 10 ml fraction. By analyzing with the native polyacrylamide gel electrophoresis, 30 kDa proteins (30 kDa) were shown to be recovered in buffer elution, vitellin in 0.2 M KC1 and egg-specific protein in 0.5 M KCI. 0
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Fraction number Fig. 3. Separation of 30 kDa protein components by the second DEAE-cellulose column chromatography. 30 kDa protein fractions from the first column were dialyzed against buffer C and applied to a DEAE-cellulose column (1.3 (i.d.) × 25 cm) pre-equilibrated with buffer C. After washing with the same buffer, the column was eluted with a linear gradient of 0-0.1 M KCI at a flow rate of 30 m l / h . The fraction size was 5 ml. Peaks 1, 2 and 3 were identified as 30 kDa protein-I, -2 and -3 by polyacrylamide gel electrophoresis (see Fig. 1).
0
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2o 10 30 Retention time(min) Fig. 4. Elution profile of vitellin (Vtn), egg-specific protein (ESP) and 30 kDa proteins on HPLC equipped with TSK gel-G 3000 SW column (7.5 mm (i.d.)x60 cm). 50 #1 of prepurified sample (about 1.5 mg protein) from DEAE-cellulose column chromatography was injected at once and eluted with buffer Q at a flow rate of 1 m//min. Each peak was collected and analyzed by native- and SDS-polyacrylamide gel electrophoresis. The peaks were identified as vitellin, eggspecific protein and 30 kDa protein-I, -2 and -3. 0
polyacrylamide gel electrophoresis (Fig. 1). From these results, vitellin, egg-specific protein and 30 kDa protein-l, -2 and -3 were conceived to be purified to homogeneity by the HPLC.
Molecular properties The apparent molecular masses of native forms of vitellin, egg-specific protein and 30 kDa proteins were estimated by chromatographies using Sephacryl S-300, Sephadex G-200 and TSK gel-G 3000 SW. They gave a linear relationship between distribution coefficients (K,v) and the log molecular weights of five marker proteins, from which average molecular masses were estimated as follows: 420 kDa for vitellin, 225 kDa for egg-specific protein, 31.5 kDa for 30 kDa protein-I, 30.0 kDa for 30 kDa protein-2 and 28.5 kDa for 30 kDa protein-3 (Table I). Subunit structures of each protein were estimated using six standard proteins on SDS-polyacrylamide gel electrophoresis under the various gel acrylamide concentrations (Fig. 1B; Table I). Vitellin consisted of two subunits with the respective molecular masses of 178 and 43 kDa, egg-specific protein of two subunits of 72 and 64 kDa, and 30 kDa protein-1 with one 32.0
432 TABLE I
A
MOLECULAR MASSES (IN kDa) AND SUBUNIT COMPOSITIONS OF VITELLIN, EGG-SPECIFIC PROTEIN AND 30 kDa PROTEINS PURIFIED FROM THE NEWLY LAID EGGS Native molecular masses were estimated by chromatographies on a Sephacryl S-300, b Sephadex G-200 and c TSK gel-G 3000 SW, and on a native polyacrylamide gel electrophoresis using standard marker proteins: thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), bovine serum albumin (67 kDa). Subunit molecular weights were determined by SDS-polyacrylamide gel electrophoresis using 8.5%, 10% and 11% gels along with the marker proteins, phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa) and a-lactalbumin (14.4 kDa). Molar ratios of subunits were estimated from densitometric analysis. The values represent means from three determinations.
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Native Subunit molecular number molecular molar mass mass ratios Vitellin
430 a 410 a
2
Egg-specific protein 220 a 226 b 230 c
2
30 kDa protein-1 -2 -3
1 1 1
31.5 b,c 30.0 b,c 28.5 b.o
178 43 72 64
1
1.2 2.01 1
32.0 31.0 29.5
k D a subunit, 30 k D a protein-2 with one 31.0 k D a s u b u n i t a n d 30 k D a protein-3 with one 29.5 k D a subunit. A densitometric analysis gave information on p r o t e i n structure: vitellin was a tetramer consisting of two heavy a n d two light subunits, egg-specific p r o t e i n was a trimer of two heavy s u b u n i t s a n d one light s u b u n i t a n d each 30 k D a protein was a m o n o m e r . For vitellin a n d 30 k D a proteins, these structural data largely confirmed the results reported previously [10,12]. However, the present results on egg-specific p r o t e i n did not c o n f i r m the s u b u n i t structure proposed by Irie a n d Y a m a s h i t a [6], who estimated it to be a dimer (molecular mass of 125 kDa) of 55 k D a subunits. W h e n the prepurified egg-specific protein was i n c u b a t e d in vitro in buffer B for 10 h at 3 7 ° C , 225 k D a egg-specific protein almost disappeared a n d was converted
a
b
a
b
Fig. 5. Conversion of native 225 kDa egg-specific protein to 125 kDa egg-specific protein by in vitro incubation. The prepurified egg-specific protein (about 50 /zg) from DEAE-cellulose column chromatography was incubated in vitro at 37 oC for 10 h. The non-incubated (a) and incubated samples (b) were subjected to a native polyacrylamide gel electrophoresis (A) and SDS-polyacrylamidegel electrophoresis (B). Molecular weights were estimated by referring to the mobility of the marker proteins.
into 125 k D a egg-specific protein (Fig. 5A). Similarly, the 72 k D a a n d 64 k D a s u b u n i t s were transformed into 55 k D a s u b u n i t (Fig. 5B). These results suggest that the 125 k D a egg-specific protein with 55 k D a s u b u n i t is a partially degraded product of native egg-specific protein. Thus, a rapid purification by H P L C could m a i n t a i n the eggspecific p r o t e i n in its native form.
Amino acids, lipids and carbohydrates A m i n o acid compositions of yolk proteins are shown in T a b l e II. N o n e of the proteins were of u n u s u a l c o m p o s i t i o n a n d all were enriched in glutamate/glutamine and aspartate/asparagine a n d low in methionine. After extraction with chloroform a n d methanol, the lipids were weighed a n d f o u n d to total approx. 4.7% b y weight of vitellin, 3.5% of egg-specific protein a n d 0.7% of 30 k D a
433 TABLE II CHEMICAL COMPOSITIONS OF VITELLIN, EGG-SPECIFIC PROTEIN AND 30 kDa PROTEINS P U R I F I E D FROM THE NEWLY LAID EGGS The results on amino acid compositions were from a typical experiment. Values on lipids and carbohydrates represent means from three determinations. S.D. < 10%. Vitellin
Amino acid (mol/1000mol) * Asp/Apn Thr Ser Glu/Gln Pro Gly Ala Val Met Ile Leu Tyr Phe His Lys Arg
117 45 44 146 46 51 75 64 20 54 74 34 52 40 79 54
Egg-specific protein
30 kDa protein 1
2
3
155 34 48 134 27 55 83 79 23 54 85 14 18 75 85 29
172 28 15 114 19 82 64 76 15 62 89 47 51 21 88 55
165 16 14 97 29 75 74 93 22 46 95 67 39 19 86 59
154 30 23 130 20 114 77 71 12 57 80 31 44 21 88 45
Lipids(%)
4.7
3.5
0.7 **
Carbohydrates(%)
1.5
2.0
2.2 **
. Cysteine and tryptophan were not determined. ** Determined on total 30 kDa proteins.
proteins (Table II). In all these proteins carbohydrates comprised about 2% by weight of total protein (Table II). The present results were fairly similar to those reported previously [6,10,12].
cross-reactivity with each other, suggesting that the 30 kDa proteins shared a common antigenicity. The native egg-specific protein cross-reacted
Immunologicalproperties The specific antiserum to each purified protein was applied to a double diffusion test to correlate the antigenicity of vitellin, egg-specific protein and 30 kDa proteins (Fig. 6-1). In some experiments, 125 kDa egg-specific protein and its antiserum, which were provided by Irie and Yamashita [6], were also used to observe the difference in antigenicity between native egg-specific protein and 125 kDa egg-specific protein (Fig. 6-2). As shown in Fig. 6-1, vitellin, egg-specific protein and 30 kDa proteins did not cross-react with each other and produced a single precipitin line against their respective antisera. Although the data are not given here, the anti-30 kDa protein sera showed
Fig. 6. Ouchterlony immunodiffusion test for puriifed 30 kDa proteins, egg-specific protein and vitellin. In panel 1, the specific antiserum raised against total 30 kDa proteins (A), egg-specific protein (B) and vitellin (C) was allowed to react with 30 kDa proteins (a), native (225 kDa) egg-specific protein (b) and vitellin (c). In panel 2, the specific antiserum against native egg-specific protein (B) and 125 kDa egg-specific protein (B l) was diffused against native egg-specific protein (b) and 125 kDa egg-specific protein (bt). In both cases, about 40 #1 antiserum and 170 #g purified protein were applied to each well and the diffusion was allowed to continue for 2 days at room temperature.
434 with the antiserum against 125 k D a egg-specific protein and vice versa, showing that these eggspecific proteins are immunologically indistinguishable when a polyclonic antibody is used. Thus, these antisera could be used for the direct quantification of these proteins from crude extracts.
Titre changes of yolk proteins during oogenesis and embryogenesis The developmental changes in vitellin, eggspecific protein and 30 k D a proteins throughout oogenesis and embryogenesis were determined by rocket immunoelectrophoresis using the same egg extracts (Fig. 7). The present conditions gave a linear relationship between the amounts of proteins (0.5-10 /~g/well) applied and peak heights (data not shown). During oogenesis, vitellin and 30 k D a proteins increased more rapidly from the m o m e n t of vitellogenesis and attained a half-maxi m u m value on day 3, followed by a slow accumu-
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lation. In contrast, egg-specific protein began to increase after a lag period of 2 days and then rose steeply towards egg maturation, reaching a halfm a x i m u m a m o u n t on day 5. In mature eggs from ovaries of newly emerged adults, vitellin, eggspecific protein and 30 k D a proteins accounted for about 40%, 25% and 35%, respectively. The eggs developed in male hosts (referred to as male eggs) were deficient in vitellin but retained the ability to complete their embryogenesis to larvae [14]. To see whether male eggs can accumulate egg-specific protein and 30 k D a proteins in the same way as the normal female eggs, the extracts of mature male eggs were subjected to immunochemical titration (Table III). Vitellin of male eggs was less than 5% of that of female eggs, but egg-specific protein and 30 k D a proteins were found at comparable levels. A b o u t 10% of the increase in protein content in newly laid (day 0) eggs seemed to be due to the loss of the ovarian sheath by oviposition. The titre changes in yolk proteins were followed throughout embryonic life (Fig. 7). During the first 6 days, when embryonic differentiation was completed, a slight and continuous decrease was observed in egg-specific protein, but vitellin and 30 k D a proteins remained almost unchanged. During larval differentiation after day 6, egg-specific protein decreased precipitously and remained at a trace level in the newly hatched larvae. Vitellin abruptly began to decrease at this stage and precipitously declined, but about 30% of the initial levels remained unused at larval hatching. On the other hand, 30 k D a proteins were persistent for more
10
Fig. 7. Changes in titres of 30 kDa proteins, egg-specific protein and vitellin estimated by rocket immunoelectrophoresis during oogenesis and embryogenesis. Ovaries were prepared every day throughout pupal adult development to 1 day before adult emergence (day 9). The eggs laid within 3 h were pooled and incubated at 25°C in order to initiate their embryonic development synchronously. On day 1 HCI treatment was carried out to prevent diapause initiation. Larval hatching occurred on day 10. Yolk proteins were extracted with buffer A from about 200 mg of ovaries or eggs and the extracts (about 50 /~g protein) were subjected to the rocket immunoelectrophoresis which was performed using 1% agarose gel plates containing 0.8% of each antiserum. Each point represents the mean from three independent determinations with + S.D. shown by vertical lines.
TABLE 1II COMPARISON OF YOLK PROTEINS IN EGGS DEVELOPED IN FEMALE AND MALE HOSTS Male eggs were obtained by transplanting ovarian discs into male larvae [14]. Total protein content was determined on crude yolk extracts by the method of Lowry et al. [23].'Values are means of three determinations. S.D. < 10%. Yolk protein
Eggs developed in
(/~g/egg)
females
Vitellin Egg-specific protein 30 kDa proteins Total proteins
19.0
1.0
12.5 17.0 52.1
13.0 17.4 33.2
males
435 longer, and more than 5070 was not used and carried over to the hatched larvae. These results clearly demonstrated that the metabolic fates of yolk proteins during embryogenesis were different from protein to protein. Discussion
By the combination of ion exchange column chromatography and HPLC on a molecular sieving column, each yolk protein was purified to homogeneity from the same extracts of silkworm eggs (Figs. 1, 2 and 3). The physical and chemical properties of vitellin are comparable with those characterized previously [10], although the purification procedures are quite different. The 30 kDa proteins were first purified as three main components from mature eggs of silkworms. The molecular properties of these proteins were similar to those purified from larval hemolymph of silkworms [12], suggesting that no chemical modification occurred during the transport processes from hemolymph to oocytes. Egg-specific protein purified in the present experiment (Table I; Fig. 4) exhibited some different molecular weights and subunit structure compared to that purified previously [6]. This discrepancy is due to the purification procedures employed. In the previous experiments [6], the final purification was done by a Sephacryl S-200 column which was developed at room temperature (25°C) overnight, because of the more reduced adsorption onto the column and the better separation at 25°C than at 4°C. The present purification was also performed at 25 °C, but HPLC took less than 20 min for the separation of egg-specific protein (Fig. 3). Thus, such a short exposure to a high temperature seems to prevent the partial degradation of egg-specific protein. Indeed, the prepurified egg-specific protein after a DEAE-cellulose column chromatography is partially degraded into 125 kDa egg-specific protein with a 55 K subunit when incubated in vitro at 37°C (Fig. 5). Further, 125 kDa eggspecific protein appeared as a main product of egg-specific protein at the middle stages of embryogenesis (unpublished data). Thus, 125 kDa egg-specific protein is assumed to be an intermediate of the native egg-specific protein degradation by endogenous proteinase(s).
Yolk proteins of newly laid eggs of silkworms have been shown to consist of three different kinds of proteins: vitellin, egg-specific protein and 30 kDa proteins, which account for more than 90% of total yolk proteins (Table III). Such a yolk protein system is quite different from that of many other insects, where the system is more simple and vitellin accounts for 8070 or more [1], whereas the more complexed figures are noted in yolks of some lepidopterous insect eggs. In Hyalophora cecropia, vitellin accounts for less than 60% of total proteins, and the remaining consists of lipophorin (approx. 2370), paravitellin (approx. 13%), arylphorin (approx. 4.570) and microvitellin (approx. 4.570) [24]. The mature eggs of Plodia interpunctella contain two major yolk proteins, vitellin and yolk protein (Yp), at the almost same ratios [8]. Although the nomenclature has not yet been established for yolk proteins other than vitellin, paravitellin in H. cecropia eggs [24] seems to correspond in its biological origin and molecular properties to egg-specific protein in B. mori [6[ and yolk proteins (Yp 1 and Yp 2) in P. interpunctella [8]. In silkworms [25] as well as in some insects [1,4], vitellin is derived from hemolymph vitellogenin which is synthesized and released from the fat body of female animals only. Similarly, 30 kDa proteins are synthesized and released from the fat body of both sexes and sequestered by developing oocytes [12], while egg-specific protein is synthesized in follicle cells and is transported into oocytes [6]. These proteins are supposed to be taken up by an endocytosis process through the selective receptors of oocyte membranes [7,26]. The accumulation pattern of yolk proteins during oogenesis (Fig. 7) suggests that proteins could not interact with each other for the incorporation processes proposed for paravitellin of H. cecropia [27], but the degrees of accumulation are under the control of the developmental program of ovaries/oocytes. In the yolk granules which were isolated by a sucrose density-gradient centrifugation, vitellin, egg-specific protein and 30 kDa proteins were found at the same levels as in soluble fractions of eggs (unpublished data). Thus, the yolk proteins are finally assembled together into yolk granules in oocytes [24,28]. I n the mature male eggs which are deficient in vitellin (Table III), yolk granules
436
are found as in the normal eggs developed in female hosts (unpublished data), suggesting that vitellin is not essential for the organization of yolk granules [14]. Although the function of 30 kDa proteins on yolk formation remains unknown, egg-specific protein seems to be important in granule formation, since the appearance of the granules in oocytes [29] is only well correlated with the accumulation profile of egg-specific protein (Fig. 7). The different decreases of yolk proteins in the course of embryogenesis and the subsequent larval differentiation (Fig. 7) leads us to conclude that the utilization of each yolk protein is closely correlated with the developmental events of embryos. The early and complete exhaustion of egg-specific protein implies that it serves as the more essential source for embryonic growth and larval differentiation. This is partly supported by the experiments on paravitellin of H. cecropia [24] and Yp 2 and Yp 4 proteins of P. interpunctella [8]. Although vitellin is a predominant component of yolk proteins (Table I), about 30% of the initial content remained unused at the larval hatching (Fig. 7), suggesting that vitellin is an additional source of energy as well as for protein synthesis in embryos. This point has been conceived by the previous experiments on male egg systems [14]. The more delayed and partial utilization of 30 kDa proteins is suggestive that these proteins are also provided as stored reserves for the newly hatched larvae. Accordingly, vitellin, egg-specific protein and 30 kDa proteins in silkworm eggs are conceived to have specialized functions in the physiological events during embryonic life.
Acknowledgments We wish to thank Professor S. Kawase and Dr. M. Kobayashi for their continuous encouragement. We are also grateful to Drs. K. Imai and T. Sasaki for amino acid analysis. The present work was supported in part by a Grant-in-Aid for Scientific Research (60304009) from the Ministry of Education, Science and Culture of Japan.
References 1 Engelman, F. (1979) Adv. Insect Physiol. 14, 49-108 2 Tata, J.R. and Smith, D.F. (1979) Recent Prog. Horm. Res. 35, 47-90 3 Hagedorn, H.H. and Kunkel, J.C. (1979) Annu. Rev. Entomol. 24, 475-505 4 Kunkel, J.G. and Nordin, J.H. (1985) in Comprehensive Insect Physiology, Biochemistry and Pharmacology (Kerkut, G.A. and Gilbert, L.I, eds.), Vol. 1, pp. 83-111, Pergamon Press, Oxford 5 Irie, K. and Yamashita, O. (1980) J. Insect Physiol. 26, 811-817 6 Irie, K. and Yamashita, O. (1983) Insect Biochem. 13, 71-80 7 Telfer, W.H., Woodruff, R.I. and Huebner, E. (1981) Am. Zool. 21,675-686 8 Shirk, P.D., Bean, D., Millemann, A.M. and Brookes, V.J. (1984) J. Exp. Zool. 232, 87-98 9 Harnish, D.G. and White, B.N. (1982) J. Exp. Zool. 200, 11-19 10 Izumi, S., Tomino, S. and Chino, H. (1980) Insect Biochem. 10, 199-208 11 Takesue, S., Onitake, K., Keino, H. and Takesue, Y. (1983) Roux's Arch. Dev. Biol. 192, 113-119 12 Izumi, S., Fujie, J., Yamada, S. and Tomino, S. (1981) Biochim. Biophys. Acta 670, 222-229 13 Ono, S., Nagayama, H. and Shimura, K. (1975) Insect Biochem. 5, 313-329 14 Yarnashita, O. and Irie, K. (1980) Nature (Lond.) 283, 385-386 15 Yamashita, O. and Hasegawa, K. (1974) J. Insect Physiol. 20, 1749-1760 16 Yamashita, O. (1984) Adv. Invertebr. Rep. 3, 251-258 17 Davis, B.J. (1964) Ann. N.Y. Acad. Sci. 131,404-427 18 Laemmli, U.K. (1970) Nature (Lond.) 227, 680-685 19 Ouchterlony, O. and Nilson, L.A. (1973) in Handbook of Experimental Immunology (Weir, D.M., ed.), 2nd Edn., pp. 19.1-19.39, Blackwell Scientific Publications, Oxford 20 Laurell, C.B. (1966) Anal. Biochem. 15, 45-52 21 Folch, J., Lees, M. and Sloane-Stanley, G.H. (1957) J. Biol. Chem. 226, 497-509 22 Mokrasch, L.C. (1954) J. Biol. Chem. 208, 55-59 23 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 24 Telfer, W.H., Rubenstein, E. and Pan, M.L. (1981) in Regulation of Insect Development and Behavior (Sehnal, F., Zabza, A., Menn, J.J. and Cymborowski, B., eds.), pp. 637-64, Scientific Papers of the Institute of Organic and Physical Chemistry of Wroclaw Technical University 25 Miwa, H. and Yamashita, O. (1985) Int. J. Invertebr. Reprod. 8, 193-196 27 Bast, R.E. and Telfer, W.H. (1976) Dev. Biol. 52, 83-97 28 Yamauchi, H., Kurihara, M. and Miya ,K. (1981) J. Fac. Agric. Iwate Univ. 15, 153-174. 29 Otsuki, Y. (1965) J. Fac. Text. Fibres Kyoto Univ. Ind. Arts and Text. Fibres 4, 314-344
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Elsevier BBA 22354
Characterization of vitellin, egg-specific protein and 30 kDa protein from Bombyx eggs, and their fates during oogenesis and embryogenesis Jiang Zhu *, Leslie S. Indrasith and Okitsugu Yamashita ** Laboratory of Sericultural Science, Faculty of Agriculture, Nagoya University, Chikusa, Nagoya 464 (Japan) (Received January 20th, 1986)
Key words: Vitellin; Egg-specific protein; 30 kDa protein; Antigenicity; Oogenesis; Embryogenesis; (B. mori egg)
Three major yolk proteins, vitellin, egg-specific protein and 30 kDa proteins, were purified from the same extracts of Bombyx mori eggs by high-performance liquid chromatography on a molecular sieving column. Each preparation was judged to be homogeneous by polyacrylamide gel electrophoresis. The subunit structure was estimated to be as follows: vitellin is a tetramer with a molecular mass of 420 kDa, consisting of two heavy subunits (178 kDa) and two light subunits (43 kDa); egg-specific protein is a trimer (225 kDa) of two heavy subunits (72 kDa) and one light subunit (64 kDa); 30 kDa proteins are a mixture of three monomers (I, 2 and 3) consisting of respective subunit molecular masses of 32.0, 31.0 and 29.5 kDa. The three yolk proteins contained the usual amino acids together with various lipids and carbohydrates. Antisera to each protein did not cross-react. The titration of vitellin, egg-specific protein and 30 kDa proteins on rocket immunoelectrophoresis showed a differential accumulation pattern during the course of oogenesis. In newly laid eggs, vitellin, egg-specific protein and 30 kDa proteins accounted for approx. 40%, 25% and 35%, respectively, in weight. The eggs developed in male hosts after implantation of ovary discs were deficient in vitellin but contained egg-specific protein and 30 kDa proteins at comparable levels to the normal female eggs. During embryogenesis, egg-specific protein was rapidly and completely utilized. Approx. 35% vitellin and 50% 30 kDa proteins remained unused and were carried over to the hatched larvae. Such accumulation and utilization of yolk proteins are correlated with the fates of the proteins during oogenesis and embryogenesis of B. mori.
Introduction
The eggs of oviparous animals are generally characterized by a high accumulation of a single species of the protein vitellin [1,2]. In many insect species, vitellin accounts for more than 80% of the total yolk proteins, and is considered to be the sole storage reserve for subsequent embryogenesis [1,2,4]. In eggs of some lepidopteran insects, in* Present address: Suzhou Sericultural College, Suzhou, China. ** To whom correspondence should be addressed. Abbreviation: PMSF, phenylmethylsulfonyl fluoride.
cluding silkworms, vitellin comprises only about half of the total yolk proteins; the yolk contains significant amounts of other kinds of proteins [5-81. There are many reports on the physicochemical properties of insect vitellins, and they are now classified into three groups depending on the molecular structure [9]. Silkworm vitellin was purified from mature eggs and characterized to be a vitellin belonging to group 1 which consists of two heavy subunits and two light subunits [10]. Recently, Takesue et al. [11] reported a higher molecular weight for silkworm vitellin, but did not give any information on its subunit structure. The
0304-4165/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
428 second major yolk protein group of silkworm eggs consists of the non-sex-linked serum proteins with molecular masses of approx. 30 kDa [12] (referred to as 30 kDa proteins). These proteins are produced in the fat body and released into the hemolymph towards larval maturation and are finally sequestered into developing oocytes [5,10,12]. Izumi et al. [12] purified 30 kDa proteins from the hemolymph of mature silkworm larvae and characterized their molecular properties. However, no attempt was made to purify and characterize them from eggs, although their behavior on polyacrylamide gel electrophoresis seemed to be quite similar. The third main protein of silkworm eggs is the so-called egg-specific protein, which is produced by the ovary itself and accumulates especially in developing oocytes [6,13]. This protein was purified from eggs, and its structural characteristics were examined by Irie and Yamashita [6]. The more important physiological events during oogenesis and embryogenesis are dependent on this yolk protein [6,14]. To consider further their physiological roles, it is desirable to identify each protein in the same egg sample. In the present study, we purified sequentially 30 kDa proteins, vitellin and eggspecific protein from the same crude extracts and characterized their molecular properties. Further, using specific antisera, the developmental changes in the protein titres were specified on the same materials throughout oogenesis and embryogenesis. Materials and Methods
Preparation of hemolymph, ovaries and eggs. Three hybrid races (Shunrei x Shogetsu, Kinshu x Showa and Nichi 106 x Daizo) of the silkworm, Bombyx mori, were used and their embryonic development was progressed at a high temperature (25°C) to obtain diapause eggs at the next generation. The larvae were reared on fresh mulberry leaves or an artificial diet (Takeda Chemical Co., Tokyo) at 25 o C. The larval life took 27 days and adults emerged 10 days after pupal ecdysis. In some experiments, ovarian discs were transplanted into male 5th instar larvae to obtain vitellin-deficient eggs [14]. Developing ovaries were prepared daily throughout pupal-to-adult development [15].
Hemolymph was collected from male and female pharate adults of day 5, when the active vitellogenesis occurred in females. The female moths were allowed to lay eggs for a period of 3 h, and the newly laid eggs were stored at - 7 0 ° C for purification of yolk proteins or incubated at 25 °C to allow embryogenesis to occur. HC1 treatment was performed on day 1 to avoid diapause [16]. In these eggs larval hatching occurred mainly on day 10. Preparation of crude extracts. All procedures were carried out at 4°C or in ice, unless specified. Developing ovaries and eggs were homogenized in 5 vol. buffer A (50 mM phosphate buffer (pH 7.2)/0.4 M KC1/1 mM PMSF) using first a mortar and then a glass-Teflon homogenizer. The homogenate was centrifuged at 16000 x g for 20 min and the supernatant was used as the crude extract for purification and titration of yolk proteins. Hemolymph was diluted 5-fold with buffer A and processed as the ovary homogenate. DEAE-cellulose column chromatography. The crude extracts were prepared from about 20 g of eggs and dialyzed against three changes of buffer B (buffer A without KC1) overnight. The dialyzed solution was applied onto a column (2.5 (internal diameter) x 65 cm) of DEAE-cellulose (DE-23, Whatman Inc., Clifton, N J) which was preconditioned with buffer B. The column was washed with buffer B and eluted with 0.2 M KC1 and then 0.5 M KC1. Flow rate was 40 m l / h and a 10 ml fraction was collected. Proteins were monitored by absorbance at 280 nm and mobility on polyacrylamide gel electrophoresis. Each fraction was pooled and concentrated by a 60% saturation of ammonium sulfate. The fractions washed out from the first DEAE-cellulose column were dialyzed against three changes of buffer C (10 mM phosphate buffer (pH 8.0)/1 mM PMSF) and applied onto the second DEAE-cellulose column (1.3 (i.d.) x 25 cm) pre-equilibrated with buffer C. The column was developed with a linear gradient of 0-0.1 M KC1 at a flow rate of 40 m l / h . Protein fractions were pooled and concentrated by ammonium sulfate as above. High-performance liquid chromatography. Each protein sample from DEAE-cellulose column chromatographies was subjected to HPLC (Tri-
429 rotar III, Japan Spectroscopic Co., Tokyo) equipped with a molecular sieving column (7.5 mm (i.d.)x 60 cm; TSK gel-G 3000 SW, Toyo Soda Co., Tokyo). A 50/~1 sample (approx. 1.5 mg proteins) was injected at once and was eluted with buffer D (buffer B containing 0.1 M sodium sulfate) at a flow rate of 1 m l / m i n under a constant pressure of 30 k g / c m 2. Each peak was collected and concentrated using a centriflow filter (Amicon Co., Danvers, MA).
Sephadex G-200 and Sephacryl S-300 column chromatography. The purified yolk proteins were applied to a Sephadex G-200 column (1.6 (i.d.) x 100 cm) and a Sephacryl S-300 column (1.6 (i.d.) x 90 cm). The respective columns were eluted at a flow rate of 8 m l / h with 50 mM phospahte buffer (pH 7.5) containing 50 mM NaC1 and buffer B. Distribution coefficients (Kay) of proteins were determined from the void volume (V0) estimated by Blue dextran and the column volume (Vt) by tyrosine. The native molecular weights were calibrated by plotting Kav vs. log molecular weights of the standard proteins (Pharmacia Fine Chemicals, Uppsala). Polyacrylamide gel electrophoresis. Non-denatured electrophoresis of purified vitellin, eggspecific protein, 30 kDa proteins and crude extracts was carried out on a 6% polyacrylamide slab at pH 9.5 [17]. SDS-polyacrylamide gel electrophoresis was performed on 8.5%, 10% and 11.5% polyacrylamide gels containing 0.1% SDS [18]. Proteins were stained with Coomassie brilliant blue R-250 (Sigma Chemicals Co., St. Louis, MO). Relative staining intensity of bands was determined by a computing densitometer (Gelman Instrument Co., Ann Arbor, MI). Molecular weights were estimated using high- and lowmolecular-weight calibration kits (Pharmacia Fine Chemicals, Uppsala). Immunochemical analysis. Rabbits were injected subcutaneously at multiple sites with 5 m g / 5 ml of each purified protein mixed with an equal volume of Freund's complete adjuvant (Difco Laboratories, Detroit, MI), and after 30 days a booster injection was given. One week later the serum was obtained and stored at - 7 0 ° C . The anti-125 kDa egg-specific protein serum provided by Irie and Yamashita [6] was used in some experiments.
Immunodiffusion was carried out in 1.2% agar for 2 days according to the method of Ouchterlony and Nilson [19]. Rocket immunoelectrophoresis was run for 24 h at 7 mA using 1.0% agarose in 0.5 M barbital buffer (pH 8.5) containing 0.8% antiserum [20].
Determination of amino acids, lipids, carbohydrates and proteins. A lyophilized protein sample was hydrolyzed in vacuo in 6 M HCI at 165°C for 30 min. The sample was evaporated to dryness and dissolved in 0.2 M citrate buffer (pH 2.2). This solution was subjected to an amino acid analyser (JLC-68A, JEOL, Tokyo). Total lipids were extracted three times from the purified proteins (35-40 mg) with 3 ml mixture of chloroform and methanol (2 : 1, v/v) [21]. After washing with distilled water, the remaining residues were measured gravimetrically. Total carbohydrates were estimated using the anthrone method [22], and amounts were expressed as mannose equivalent. Protein content was determined by the method of Lowry et al. [23] using bovine serum albumin as a standard. Results
Identification of yolk proteins Fig. 1A shows a native polyacrylamide gel electrophoresis analysis of yolk protein extracts from the newly laid eggs along with serum proteins from male and female pharate adults. Female serum proteins (lane b) were separated into four main bands, numbered 1 to 4 in order of moving distance, but band 1 was completely deficient in male serum (lane a). Egg extracts (lane c) showed one additional main band (band 5). These tissueand sex-dependent banding patterns suggest that band 1 is from vitellogenin/Vtn [10], band 5 from egg-specific protein [6] and bands 2, 3 and 4 from 30 kDa proteins [12]. The SDS-polyacrylamide gel electrophoresis (Fig. 1B) of yolk proteins (lane c) gave seven main bands, numbered 1 to 7, and some minor bands. Bands 1 and 4 had respective molecular masses of 178 and 43 kDa (cf. Table I), and were completely deficient in male serum (lane a), thus corresponding to heavy and light subunits of vitellin [10]. Bands 2 and 3, with respective molecular masses of 72 and 64 kDa, were estimated to be
430
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B
jj
I
J
I
e
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Fig. 1. Banding patterns of proteins on native polyacrylamide gel electrophoresis (A) and an SDS-polyacrylamide gel electrophoresis (B). Protein samples (50-60 ktg) were from hemolymph of 5-day-old male pharate adults (a), hemolymph of 5-day-old vitellogenic females (b), newly laid eggs (c), purified vitellin (d), purified egg-specific protein (e), purified 30 kDa protein-1 (f), -2 (g), -3 (h). Native polyacrylamide gel electrophoresis (A) was done using 6% polyacrylamide gels and the major bands 1-5 in the migration order corresponded to vitellin, 30 kDa protein-l, 30 kDa protein-2, 30 kDa protein-3 and egg-specific protein, respectively. The polypeptide bands on SDS-polyacrylamide gel electrophoresis using 11% gels (B) were estimated as follows: band 1, heavy subunit (178 kDa) of vitellin; band 2, heavy subunit (72 kDa) of egg-specific protein; band 3, light subunit (64 kDa) of egg-specific protein; band 4, light subunit (43 kDa) of vitellin; bands 5, 6 and 7, polypeptide of 30 kDa protein-1 (32.0 kDa), -2 (31.0 kDa) and -3 (29.5 kDa), respectively.
subunits of egg-specific protein, because of their absence in serum samples (lanes a and b) [6]. Bands 5, 6 and 7, migrating with molecular masses of appro. 30 kDa, were identified as 30 k D a protein-l, -2 and -3 [12]. Consequently, the yolk of silkworm eggs consisted of the three groups of proteins, vitellin, egg-specific protein and 30 k D a proteins, which differed with regard to tissue distribution, sex-linkage and molecular weights.
Purification of yolk proteins The first DEAE-cellulose column chromatography of the egg extracts (Fig. 2) showed that not all c o m p o n e n t s of the 30 k D a proteins b o u n d to the column. Vitellin was eluted at 0.2 M KC1 and egg-specific protein at 0.5 M KC1. The separated proteins were precipitated with 60% a m m o n i u m sulfate and dissolved in the following buffer solutions: 30 k D a proteins in buffer C; vitellin in
buffer A; and egg-specific protein in buffer B. The fractions of vitellin and egg-specific protein were directly subjected to H P L C as described below. The 30 k D a protein fractions were again subjected to the second DEAE-cellulose column chrom a t o g r a p h y which was developed by a linear gradient (0-0.1 M KCI in buffer C) (Fig. 3). 30 k D a protein-1 was recovered by the buffer washing, 30 k D a protein-2 was eluted at 0.05 M KC1, and 30 k D a protein-3 at 0.08 M KC1. Each fraction was concentrated and used for the final purification using H P L C . The prepurified vitellin, egg-specific protein and 30 k D a proteins were subjected to H P L C which was developed with buffer D at a flow rate of 1 m l / m i n and were monitored at 280 nm (Fig. 4). Each protein was eluted as a single peak at the following specific retention times: 13 min for vitellin, 16 min for egg-specific protein, 21 min for 30
431 141
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160 80 120 Fraction number Fig. 2. Separation of 30 kDa proteins, vitellin (Vtn) and egg-specific protein (ESP) from the newly laid egg homogenates by the first DEAE-cellulose column chromatography. The dialyzed crude extracts from about 20 g of eggs were applied to the column (2.5 (i.d.)× 65 cm) pre-equilibrated with buffer B and washed with the same buffer. Further elution was done stepwise using the same buffer containing 0.2 M KC1 and then 0.5 M KCI. The column was developed at a flow rate of 40 m l / h with a 10 ml fraction. By analyzing with the native polyacrylamide gel electrophoresis, 30 kDa proteins (30 kDa) were shown to be recovered in buffer elution, vitellin in 0.2 M KC1 and egg-specific protein in 0.5 M KCI. 0
1
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Fraction number Fig. 3. Separation of 30 kDa protein components by the second DEAE-cellulose column chromatography. 30 kDa protein fractions from the first column were dialyzed against buffer C and applied to a DEAE-cellulose column (1.3 (i.d.) × 25 cm) pre-equilibrated with buffer C. After washing with the same buffer, the column was eluted with a linear gradient of 0-0.1 M KCI at a flow rate of 30 m l / h . The fraction size was 5 ml. Peaks 1, 2 and 3 were identified as 30 kDa protein-I, -2 and -3 by polyacrylamide gel electrophoresis (see Fig. 1).
0
i
i
i
i
i
2o 10 30 Retention time(min) Fig. 4. Elution profile of vitellin (Vtn), egg-specific protein (ESP) and 30 kDa proteins on HPLC equipped with TSK gel-G 3000 SW column (7.5 mm (i.d.)x60 cm). 50 #1 of prepurified sample (about 1.5 mg protein) from DEAE-cellulose column chromatography was injected at once and eluted with buffer Q at a flow rate of 1 m//min. Each peak was collected and analyzed by native- and SDS-polyacrylamide gel electrophoresis. The peaks were identified as vitellin, eggspecific protein and 30 kDa protein-I, -2 and -3. 0
polyacrylamide gel electrophoresis (Fig. 1). From these results, vitellin, egg-specific protein and 30 kDa protein-l, -2 and -3 were conceived to be purified to homogeneity by the HPLC.
Molecular properties The apparent molecular masses of native forms of vitellin, egg-specific protein and 30 kDa proteins were estimated by chromatographies using Sephacryl S-300, Sephadex G-200 and TSK gel-G 3000 SW. They gave a linear relationship between distribution coefficients (K,v) and the log molecular weights of five marker proteins, from which average molecular masses were estimated as follows: 420 kDa for vitellin, 225 kDa for egg-specific protein, 31.5 kDa for 30 kDa protein-I, 30.0 kDa for 30 kDa protein-2 and 28.5 kDa for 30 kDa protein-3 (Table I). Subunit structures of each protein were estimated using six standard proteins on SDS-polyacrylamide gel electrophoresis under the various gel acrylamide concentrations (Fig. 1B; Table I). Vitellin consisted of two subunits with the respective molecular masses of 178 and 43 kDa, egg-specific protein of two subunits of 72 and 64 kDa, and 30 kDa protein-1 with one 32.0
432 TABLE I
A
MOLECULAR MASSES (IN kDa) AND SUBUNIT COMPOSITIONS OF VITELLIN, EGG-SPECIFIC PROTEIN AND 30 kDa PROTEINS PURIFIED FROM THE NEWLY LAID EGGS Native molecular masses were estimated by chromatographies on a Sephacryl S-300, b Sephadex G-200 and c TSK gel-G 3000 SW, and on a native polyacrylamide gel electrophoresis using standard marker proteins: thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), bovine serum albumin (67 kDa). Subunit molecular weights were determined by SDS-polyacrylamide gel electrophoresis using 8.5%, 10% and 11% gels along with the marker proteins, phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa) and a-lactalbumin (14.4 kDa). Molar ratios of subunits were estimated from densitometric analysis. The values represent means from three determinations.
i/!~ii~i~i~'!' i~
Native Subunit molecular number molecular molar mass mass ratios Vitellin
430 a 410 a
2
Egg-specific protein 220 a 226 b 230 c
2
30 kDa protein-1 -2 -3
1 1 1
31.5 b,c 30.0 b,c 28.5 b.o
178 43 72 64
1
1.2 2.01 1
32.0 31.0 29.5
k D a subunit, 30 k D a protein-2 with one 31.0 k D a s u b u n i t a n d 30 k D a protein-3 with one 29.5 k D a subunit. A densitometric analysis gave information on p r o t e i n structure: vitellin was a tetramer consisting of two heavy a n d two light subunits, egg-specific p r o t e i n was a trimer of two heavy s u b u n i t s a n d one light s u b u n i t a n d each 30 k D a protein was a m o n o m e r . For vitellin a n d 30 k D a proteins, these structural data largely confirmed the results reported previously [10,12]. However, the present results on egg-specific p r o t e i n did not c o n f i r m the s u b u n i t structure proposed by Irie a n d Y a m a s h i t a [6], who estimated it to be a dimer (molecular mass of 125 kDa) of 55 k D a subunits. W h e n the prepurified egg-specific protein was i n c u b a t e d in vitro in buffer B for 10 h at 3 7 ° C , 225 k D a egg-specific protein almost disappeared a n d was converted
a
b
a
b
Fig. 5. Conversion of native 225 kDa egg-specific protein to 125 kDa egg-specific protein by in vitro incubation. The prepurified egg-specific protein (about 50 /zg) from DEAE-cellulose column chromatography was incubated in vitro at 37 oC for 10 h. The non-incubated (a) and incubated samples (b) were subjected to a native polyacrylamide gel electrophoresis (A) and SDS-polyacrylamidegel electrophoresis (B). Molecular weights were estimated by referring to the mobility of the marker proteins.
into 125 k D a egg-specific protein (Fig. 5A). Similarly, the 72 k D a a n d 64 k D a s u b u n i t s were transformed into 55 k D a s u b u n i t (Fig. 5B). These results suggest that the 125 k D a egg-specific protein with 55 k D a s u b u n i t is a partially degraded product of native egg-specific protein. Thus, a rapid purification by H P L C could m a i n t a i n the eggspecific p r o t e i n in its native form.
Amino acids, lipids and carbohydrates A m i n o acid compositions of yolk proteins are shown in T a b l e II. N o n e of the proteins were of u n u s u a l c o m p o s i t i o n a n d all were enriched in glutamate/glutamine and aspartate/asparagine a n d low in methionine. After extraction with chloroform a n d methanol, the lipids were weighed a n d f o u n d to total approx. 4.7% b y weight of vitellin, 3.5% of egg-specific protein a n d 0.7% of 30 k D a
433 TABLE II CHEMICAL COMPOSITIONS OF VITELLIN, EGG-SPECIFIC PROTEIN AND 30 kDa PROTEINS P U R I F I E D FROM THE NEWLY LAID EGGS The results on amino acid compositions were from a typical experiment. Values on lipids and carbohydrates represent means from three determinations. S.D. < 10%. Vitellin
Amino acid (mol/1000mol) * Asp/Apn Thr Ser Glu/Gln Pro Gly Ala Val Met Ile Leu Tyr Phe His Lys Arg
117 45 44 146 46 51 75 64 20 54 74 34 52 40 79 54
Egg-specific protein
30 kDa protein 1
2
3
155 34 48 134 27 55 83 79 23 54 85 14 18 75 85 29
172 28 15 114 19 82 64 76 15 62 89 47 51 21 88 55
165 16 14 97 29 75 74 93 22 46 95 67 39 19 86 59
154 30 23 130 20 114 77 71 12 57 80 31 44 21 88 45
Lipids(%)
4.7
3.5
0.7 **
Carbohydrates(%)
1.5
2.0
2.2 **
. Cysteine and tryptophan were not determined. ** Determined on total 30 kDa proteins.
proteins (Table II). In all these proteins carbohydrates comprised about 2% by weight of total protein (Table II). The present results were fairly similar to those reported previously [6,10,12].
cross-reactivity with each other, suggesting that the 30 kDa proteins shared a common antigenicity. The native egg-specific protein cross-reacted
Immunologicalproperties The specific antiserum to each purified protein was applied to a double diffusion test to correlate the antigenicity of vitellin, egg-specific protein and 30 kDa proteins (Fig. 6-1). In some experiments, 125 kDa egg-specific protein and its antiserum, which were provided by Irie and Yamashita [6], were also used to observe the difference in antigenicity between native egg-specific protein and 125 kDa egg-specific protein (Fig. 6-2). As shown in Fig. 6-1, vitellin, egg-specific protein and 30 kDa proteins did not cross-react with each other and produced a single precipitin line against their respective antisera. Although the data are not given here, the anti-30 kDa protein sera showed
Fig. 6. Ouchterlony immunodiffusion test for puriifed 30 kDa proteins, egg-specific protein and vitellin. In panel 1, the specific antiserum raised against total 30 kDa proteins (A), egg-specific protein (B) and vitellin (C) was allowed to react with 30 kDa proteins (a), native (225 kDa) egg-specific protein (b) and vitellin (c). In panel 2, the specific antiserum against native egg-specific protein (B) and 125 kDa egg-specific protein (B l) was diffused against native egg-specific protein (b) and 125 kDa egg-specific protein (bt). In both cases, about 40 #1 antiserum and 170 #g purified protein were applied to each well and the diffusion was allowed to continue for 2 days at room temperature.
434 with the antiserum against 125 k D a egg-specific protein and vice versa, showing that these eggspecific proteins are immunologically indistinguishable when a polyclonic antibody is used. Thus, these antisera could be used for the direct quantification of these proteins from crude extracts.
Titre changes of yolk proteins during oogenesis and embryogenesis The developmental changes in vitellin, eggspecific protein and 30 k D a proteins throughout oogenesis and embryogenesis were determined by rocket immunoelectrophoresis using the same egg extracts (Fig. 7). The present conditions gave a linear relationship between the amounts of proteins (0.5-10 /~g/well) applied and peak heights (data not shown). During oogenesis, vitellin and 30 k D a proteins increased more rapidly from the m o m e n t of vitellogenesis and attained a half-maxi m u m value on day 3, followed by a slow accumu-
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Age in days
2
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Embryogenesis
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lation. In contrast, egg-specific protein began to increase after a lag period of 2 days and then rose steeply towards egg maturation, reaching a halfm a x i m u m a m o u n t on day 5. In mature eggs from ovaries of newly emerged adults, vitellin, eggspecific protein and 30 k D a proteins accounted for about 40%, 25% and 35%, respectively. The eggs developed in male hosts (referred to as male eggs) were deficient in vitellin but retained the ability to complete their embryogenesis to larvae [14]. To see whether male eggs can accumulate egg-specific protein and 30 k D a proteins in the same way as the normal female eggs, the extracts of mature male eggs were subjected to immunochemical titration (Table III). Vitellin of male eggs was less than 5% of that of female eggs, but egg-specific protein and 30 k D a proteins were found at comparable levels. A b o u t 10% of the increase in protein content in newly laid (day 0) eggs seemed to be due to the loss of the ovarian sheath by oviposition. The titre changes in yolk proteins were followed throughout embryonic life (Fig. 7). During the first 6 days, when embryonic differentiation was completed, a slight and continuous decrease was observed in egg-specific protein, but vitellin and 30 k D a proteins remained almost unchanged. During larval differentiation after day 6, egg-specific protein decreased precipitously and remained at a trace level in the newly hatched larvae. Vitellin abruptly began to decrease at this stage and precipitously declined, but about 30% of the initial levels remained unused at larval hatching. On the other hand, 30 k D a proteins were persistent for more
10
Fig. 7. Changes in titres of 30 kDa proteins, egg-specific protein and vitellin estimated by rocket immunoelectrophoresis during oogenesis and embryogenesis. Ovaries were prepared every day throughout pupal adult development to 1 day before adult emergence (day 9). The eggs laid within 3 h were pooled and incubated at 25°C in order to initiate their embryonic development synchronously. On day 1 HCI treatment was carried out to prevent diapause initiation. Larval hatching occurred on day 10. Yolk proteins were extracted with buffer A from about 200 mg of ovaries or eggs and the extracts (about 50 /~g protein) were subjected to the rocket immunoelectrophoresis which was performed using 1% agarose gel plates containing 0.8% of each antiserum. Each point represents the mean from three independent determinations with + S.D. shown by vertical lines.
TABLE 1II COMPARISON OF YOLK PROTEINS IN EGGS DEVELOPED IN FEMALE AND MALE HOSTS Male eggs were obtained by transplanting ovarian discs into male larvae [14]. Total protein content was determined on crude yolk extracts by the method of Lowry et al. [23].'Values are means of three determinations. S.D. < 10%. Yolk protein
Eggs developed in
(/~g/egg)
females
Vitellin Egg-specific protein 30 kDa proteins Total proteins
19.0
1.0
12.5 17.0 52.1
13.0 17.4 33.2
males
435 longer, and more than 5070 was not used and carried over to the hatched larvae. These results clearly demonstrated that the metabolic fates of yolk proteins during embryogenesis were different from protein to protein. Discussion
By the combination of ion exchange column chromatography and HPLC on a molecular sieving column, each yolk protein was purified to homogeneity from the same extracts of silkworm eggs (Figs. 1, 2 and 3). The physical and chemical properties of vitellin are comparable with those characterized previously [10], although the purification procedures are quite different. The 30 kDa proteins were first purified as three main components from mature eggs of silkworms. The molecular properties of these proteins were similar to those purified from larval hemolymph of silkworms [12], suggesting that no chemical modification occurred during the transport processes from hemolymph to oocytes. Egg-specific protein purified in the present experiment (Table I; Fig. 4) exhibited some different molecular weights and subunit structure compared to that purified previously [6]. This discrepancy is due to the purification procedures employed. In the previous experiments [6], the final purification was done by a Sephacryl S-200 column which was developed at room temperature (25°C) overnight, because of the more reduced adsorption onto the column and the better separation at 25°C than at 4°C. The present purification was also performed at 25 °C, but HPLC took less than 20 min for the separation of egg-specific protein (Fig. 3). Thus, such a short exposure to a high temperature seems to prevent the partial degradation of egg-specific protein. Indeed, the prepurified egg-specific protein after a DEAE-cellulose column chromatography is partially degraded into 125 kDa egg-specific protein with a 55 K subunit when incubated in vitro at 37°C (Fig. 5). Further, 125 kDa eggspecific protein appeared as a main product of egg-specific protein at the middle stages of embryogenesis (unpublished data). Thus, 125 kDa egg-specific protein is assumed to be an intermediate of the native egg-specific protein degradation by endogenous proteinase(s).
Yolk proteins of newly laid eggs of silkworms have been shown to consist of three different kinds of proteins: vitellin, egg-specific protein and 30 kDa proteins, which account for more than 90% of total yolk proteins (Table III). Such a yolk protein system is quite different from that of many other insects, where the system is more simple and vitellin accounts for 8070 or more [1], whereas the more complexed figures are noted in yolks of some lepidopterous insect eggs. In Hyalophora cecropia, vitellin accounts for less than 60% of total proteins, and the remaining consists of lipophorin (approx. 2370), paravitellin (approx. 13%), arylphorin (approx. 4.570) and microvitellin (approx. 4.570) [24]. The mature eggs of Plodia interpunctella contain two major yolk proteins, vitellin and yolk protein (Yp), at the almost same ratios [8]. Although the nomenclature has not yet been established for yolk proteins other than vitellin, paravitellin in H. cecropia eggs [24] seems to correspond in its biological origin and molecular properties to egg-specific protein in B. mori [6[ and yolk proteins (Yp 1 and Yp 2) in P. interpunctella [8]. In silkworms [25] as well as in some insects [1,4], vitellin is derived from hemolymph vitellogenin which is synthesized and released from the fat body of female animals only. Similarly, 30 kDa proteins are synthesized and released from the fat body of both sexes and sequestered by developing oocytes [12], while egg-specific protein is synthesized in follicle cells and is transported into oocytes [6]. These proteins are supposed to be taken up by an endocytosis process through the selective receptors of oocyte membranes [7,26]. The accumulation pattern of yolk proteins during oogenesis (Fig. 7) suggests that proteins could not interact with each other for the incorporation processes proposed for paravitellin of H. cecropia [27], but the degrees of accumulation are under the control of the developmental program of ovaries/oocytes. In the yolk granules which were isolated by a sucrose density-gradient centrifugation, vitellin, egg-specific protein and 30 kDa proteins were found at the same levels as in soluble fractions of eggs (unpublished data). Thus, the yolk proteins are finally assembled together into yolk granules in oocytes [24,28]. I n the mature male eggs which are deficient in vitellin (Table III), yolk granules
436
are found as in the normal eggs developed in female hosts (unpublished data), suggesting that vitellin is not essential for the organization of yolk granules [14]. Although the function of 30 kDa proteins on yolk formation remains unknown, egg-specific protein seems to be important in granule formation, since the appearance of the granules in oocytes [29] is only well correlated with the accumulation profile of egg-specific protein (Fig. 7). The different decreases of yolk proteins in the course of embryogenesis and the subsequent larval differentiation (Fig. 7) leads us to conclude that the utilization of each yolk protein is closely correlated with the developmental events of embryos. The early and complete exhaustion of egg-specific protein implies that it serves as the more essential source for embryonic growth and larval differentiation. This is partly supported by the experiments on paravitellin of H. cecropia [24] and Yp 2 and Yp 4 proteins of P. interpunctella [8]. Although vitellin is a predominant component of yolk proteins (Table I), about 30% of the initial content remained unused at the larval hatching (Fig. 7), suggesting that vitellin is an additional source of energy as well as for protein synthesis in embryos. This point has been conceived by the previous experiments on male egg systems [14]. The more delayed and partial utilization of 30 kDa proteins is suggestive that these proteins are also provided as stored reserves for the newly hatched larvae. Accordingly, vitellin, egg-specific protein and 30 kDa proteins in silkworm eggs are conceived to have specialized functions in the physiological events during embryonic life.
Acknowledgments We wish to thank Professor S. Kawase and Dr. M. Kobayashi for their continuous encouragement. We are also grateful to Drs. K. Imai and T. Sasaki for amino acid analysis. The present work was supported in part by a Grant-in-Aid for Scientific Research (60304009) from the Ministry of Education, Science and Culture of Japan.
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