Characterization of the native vitellogenin and vitellin of the cockroach, Nauphoeta cinerea, and comparison with other species

Insect Biochem. Vol. 17, No. 2, pp. 353--365, 1987 Printed in Great Britain. All rights reserved

0020-1790/87 $3.00+0.00 Copyright © 1987 PergamonJournals Ltd

CHARACTERIZATION OF THE NATIVE VITELLOGENIN A N D VITELLIN OF THE COCKROACH, NAUPHOETA CINEREA, A N D COMPARISON WITH OTHER SPECIES H. IMBODEN,l R. KONIG,1 P. OTT, 2 A. LUSTIG,3 U. KAMPFER4 and B. LANZREIN1 aDivision of Animal Physiology, Institute of Zoology, University of Berne, Erlachstrasse 9a, CH-3012 Berne, 2Department of Biochemistry of the School of Medicine, University of Berne, Bfihlstrasse 28, CH-3012 Berne, 3Biocenter, University of Basle, CH-4056 Basle and 41nstitute of Biochemistry, University of Berne, Freiestrasse 3, CH-3012 Berne, Switzerland (Received 7 November 1985; revised and accepted 14 May 1986) Abstract--In native gradient PAGE vitellogenin of Nauphoeta cinerea showed three closely spaced bands with molecular weights (Mr) of 300,000, 314,000 and 329,000 and a broad band with a Mr of approx. 600,000, and vitellin three closely spaced bands with M# of 274,000, 293,000 and 308,000. Using the same technique vitellin of Leucophaea maderae appeared as several bands with Mrs of 270,000-350,000, Hyalophora cecropia vitellin as a single sharp band with a M r of 520,000 and Manduca sexta vitellin as three bands with MrS of 510,000, 520,000 and 545,000. Investigation of the role of the pH and the ionic strength in the analytical ultracentrifugation revealed a Mr of 244,000 for vitellin (sedimentation equilibrium method) and a s20.w-valueof 9.4 S when analyzed in PAGE buffer (pH 8.25), whereas a Mr of 491,000 and a s20.wof 15 S were found in 0.02 M sodium phosphate, 0.4 M NaCI, pH 7.5. At pH 3.6 aggregation of vitellin was observed. In sucrose density centrifugation addition of Triton X-100 altered the sedimentation coefficient from an apparent value of 15 to 8.4 S. These data suggest that the native vitellin is composed of two monomers with a Mr of 244,000 which assemble to a dimer by hydrophobic interactions. Determination of the Stokes' radius by gel permeation chromatography revealed a value of approx. 7.5 nm for both the monomeric and dimeric form of vitellin and the fractional ratio was calculated to be 1.67 for the monomer and 1.45 for the dimer indicating an elongated shape. Immunodiffusion tests showed the presence of the same two components in vitellogenin and vitellin, one component being much more abundant than the other; data from gel permeation chromatography suggest that the minor component is a smaller molecule with a Stokes' radius of 4.8 nm. Comparative analyses of the carbohydrate, lipid and amino acid composition of vitellogenin and vitellin revealed no major differences although minor differences with respect to the phospholipid composition were observed. Key Word Index: Vitellogenin, vitellin, cockroach, PAGE, analytical ultracentrifugation, sucrose density gradient, Stokes radius, Triton X-IO0, gel permeation chromatography, amino acid composition, lipid, carbohydrate, Leucophaea maderae, Nauphoeta cinerea, Manduca sexta, Hyalophora cecropia

INTRODUCTION Vitellogenins are female-specific proteins which are synthesized extraovarially and become the major egg yolk proteins, the vitellins. These are thought to be the nutritive source of amino acids, lipids, carbohydrates and phosphorus for the developing embryo. The physico-chemical properties of vitellogenins and vitellins have been investigated in several species (for reviews see Engelmann, 1979; Hagedorn and Kunkel, 1979) and it is apparent that they are high molecular weight lipoglycoproteins. In many insect species, including cockroaches, vitellogenins are synthesized in the fat body under the control of juvenile hormone, released into the haemolymph and taken up selectively by the maturing oocyte against a concentration gradient (see Engelmann, 1979, 1983). It is our aim to elucidate the process of vitellogenin incorporation and vitellin formation. F o r this purpose it is important to know the physicochemical

properties of these proteins. In our experimental insect, the ovoviviparous cockroach Nauphoeta cinerea, we recently presented evidence for the existence of specific vitellogenin binding sites on the membranes of oocytes at the stage of vitellogenesis (K6nig and Lanzrein, 1985). Here we describe the characterization of the vitellogenin and vitellin of this species. In view of the lipoglycoprotein nature and the pH and ionic strength dependent solubility and aggregation properties of these proteins (Brookes and Dejmal, 1968; Dejmal and Brookes, 1972) we used several methods and various buffers for determining their molecular weight and sedimentation constant. Here we present data obtained by immunodiffusion, different types of native polyacrylamide gel electrophoresis (PAGE), analytical ultracentrifugation (sedimentation velocity and sedimentation equilibrium), sucrose density gradient centrifugation in the presence and absence of Triton X-100 and gel permeation chromatography. We also present data on the anal-

353

H. IMBODEN et al.

354

ysis of the contents and composition of carbohydrates, lipids and amino acids. In addition we include a direct comparison of the Nauphoeta cinerea vitellin with the vitellins of another cockroach species (Leucophaea maderae) and two lepidopterous species (Hyalophora cecropia and Manduca sexta) using native gradient P A G E . MATERIALS AND METHODS

Insects Nauphoeta cinerea and Leucophaea maderae were kept at 26°C and 60% r.h. on dog flakes and water at a photoperiod of 12hr. Under these conditions oocyte maturation lasts 12-13 days for Nauphoeta cinerea and 22-26 days for Leucophaea maderae. Pupae of Hyalophora cecropia were kindly given to us by Dr W. Telfer (University of Pennsylvania, Philadelphia) and pupae of Manduca sexta by Dr R. Ziegler (Freie Universit,~t, Berlin).

Chemicals All chemicals were of analytical grade and were purchased from Merck-Schuchardt Co. if not otherwise stated. Substances for electrophoresis were obtained from BioRad.

Collection of hemolymph and oocytes Insects were immobilized on ice for 1 hr. They were then injected with 50#1 of insect Ringer solution, pH7.5, and after another 30 min on ice, the insects were bled by cutting away one leg. The hemolymph was then centrifuged for 20 rain at 10,000g at 4°C and the supernatant was diluted with phosphate buffered saline (PBS, 0.02 M sodium phosphate, 0.4 M NaC1, 0.02% NAN3, pH 7.5). In experiments to determine the chemical composition of vitellogenin, the insects were injected with 200 #1 of ice-cold Ringer solution pH 7.5 containing I mM phenylmethylsulphonylfluoride (PMSF) and the hemolymph was centrifuged for I hr at 3000 g in a Mistral 2L refrigerated centrifuge at 4°C after which the supernatant was collected. Oocytes were dissected from 10-11-day-old Nauphoeta cinerea, 20-24-day-old Laucophaea maderae (oocyte length 4 nun), pupae.ofHyalophora cecropia at the stage ofchorion formation or 2-day-old-adult females of Manduca sexta. Oocytes were then very gently homogenized in PBS and centrifuged at 10,000g (20 rain, 4°C).

Purification of vitellogenin and vitellin For isolation of viteUogenin and vitellin the DEAEchromatography method of Buschor and Lanzrein 0983) was used with the following minor modifications: the hemolymph supernatant, hereafter referred to as hemolymph, was dialyzed against 0.04 M instead of 0.02 M sodium phosphate buffer 0.15 M NaC1, 0.02% NAN3, pH 6.5, which was also the starting buffer, and the column (DEAE-Sepharose CL-tB from Pharmacia) was 1.6 x 30 cm long instead of 1.6 x 12 cm. Vitellogenin was eluted using a gradient of increasing NaC1. The fractions containing vitellogenin (as verified by Ouchterlony immunodiffusion) were pooled and dialyzed against 0.04M phosphate, 0.5 M NaC1, 0.02% NAN3, pH 7.0. Approximately 20-30 mg of the purified vitellogenin and viteUin was then applied to an affinity column containing 200 nag of IgG from an antiserum against male cockroach hemolymph covalently coupled to 15 ml Sepharose 4B (Pharmacia) at a flow-rate of 5 nil/hr. The column was washed with the same buffer and the eluate was collected and concentrated by centrifugation in Centriflo membrane cones CF 25 (Amicon Corp.) at 800 g in a Mistral 2L refrigerated centrifuge. Yield and degree of vitellogenin purification are summarized in Table I and analysis of the purity of the fractions by PAGE (data not shown) was in agreement with earlier findings of Buschor and Lanzrein (1983), who observed that vitellogenin appears electrophoretieally pure after DEAE-chromatography. Nevertheless the ratio of vitellogenin relative to the total protein content increased after affinity chromatography (Table 1), indicating an additional purification by this method. We thus used affinity chromatography purified vitellogenin and vitellin for all chemical analyses as well as for molecular weight determinations in slab gels using various concentrations of PA (Ferguson plot).

Determination of concentrations of vitellogenin, vitellin and other proteins ViteUogenin and vitellin were quantified by rocket immunoelectrophoresis as described by Busehor and Lanzrein (1983) with a DEAE-purified vitellogenin preparation of E2s0nm~- 1.0 as a standard, using an extinction coefficient of E2s0nm.~o/.= 6.25. Total protein concentrations were determined by the method of Bradford (1976) using bovine serum albumin (Bio Science) as a standard. At least two aliquots of each sample were measured in triplieate.

Preparation of antisera

Polyacrylamide gel electrophoresis (PAGE) and application for molecular weight determination

Antisera against female hemolymph prepared by G. Biihlmann (see Biihlmann, 1976) were rendered female specific by precipitation with male hemolymph or else an antiserum against DEAE purified vitellogenin was used (see Buschor and Lanzrein, 1983). For the preparation of antisera against male hemolymph proteins, hemolymph of 10-day-old males was dialyzed against saline and then 1 ml of the diaiysate containing 1.5 mg of protein was emulsified with an equal volume of Freund's complete adjuvant (Difco) and injected i.m. into rabbits six times at intervals of 2 weeks. IgG was isolated using the method of Harboe and Ingild (1973).

For identification and molecular weight determination of vitellogenin and vitellin one method applied was native gradient PAGE. Gel tubes (6 mm dis, 9 cm long) with a linear gradient of 3.8 to 18.9% acrylamide and 2.2% cross-linking agent were cast according to Margolis and Kenfick (1968) with a home-made apparatus. The buffer system of Davis (1964) was used and nine tubes were prepared simultaneously. After polymerization the gels were covered with a layer of 400/~1 of a 3% spacer gel according to Davis (1964). As molecular weight standards the electrophoresis calibration kit of Pharmacia was used, Five micro-

Table 1. Yield and degree of vitellogeninpurification Vitellogenin Total protein~ Degree of Purification step Units* Yield (%) (mg) purification:~ Female hemolymph 65.3 100 328.6 1.00 DEAE-chromatography 54.0 82 62.8 4.33 Anti-male IgG affinity-chromatography 39.9 61 31.5 6.38 *Determined using rocket immunoclectrophoresis;relative to a vitellogeninstandard of 1.0 A280/ml. ')'Determined using the Bradford protein assay with BSA as a standard. :~Relativeto the ratio of vitellogeninto total protein in the crude female hemolymph.

Native vitellogenin and vitellin of N. cinerea grams of each standard and 15-25/zg protein were applied. Electrophoresis was carried out for 20-23 hr with a constant voltage of 165 V at 4°C. After electrophoresis proteins were fixed in 10% sulfosalicylic acid for 30rain and stained in acetic acid-methanol-water (2:5:13, v/v/v) with 0.1% Coomassie blue R-250 overnight at 40°C with shaking. Destaining was performed in the same solution without Coomassie blue. The logarithm of the molecular weight of standard proteins was plotted versus the migration distance from the spacer-gel junction (Lorentz, 1976) and the resulting line was used to determine the molecular weights of vitellogenins and vitellins. As an alternative method to determine the molecular weight of vitellogenin, electrophoresis was carried out according to Davis (1964) in slab gels with a total acrylamide concentration of 5, 6.2, 6.9, 7.7, 8.5, 9.2, 10.0 and 10.8%. The degree of cross-linking was kept at 2.6% in each gel. The same molecular weight standards as mentioned above were used. To obtain a better separation of high molecular weight proteins, electrophoresis was continued after the dye front had left the gel. Total electrophoresis time was 14 hr at a constant voltage of 150 V. After electrophoresis, gels were fixed and stained as described above. The molecular weight was determined using the method of Hedrick and Smith (1968), whereby the results were expressed as a ratio of protein mobility relative to the mobility of lactate dehydrogenase.

Gel permeation chromatography A Sephacryl S-300 superfine (Pharmacia) column,. 132.5 x 1.5 cm was used at a flow rate of 12 ml/hr at 19°C. The eluate was monitored at 280 nm and the fractions were collected for further analysis with immunodiffusion or rocket immunoelectrophoresis. For calibration of the column, the gel filtration calibration kit from Pharmacia was used containing thyroglobulin, ferritin, catalase and aldolase.

355

hydrolysis of 4 to 7 nag of protein in sealed tubes with 2 ml of I N H2SO4 for 8 hr in a boiling water bath, hydrolysates were freed of charged substances by passage through anion and cation exchange resins, and the individual sugars were separated by paper chromatography (Spiro, 1966). After completion of the chromatography, the paper was dried and stained with aniline phthalate (Partridge, 1949). For quantitative analysis of the individual sugars, only the strips containing the standard mixtures were stained, whereas the rest of the paper sheet was cut in 1 era-zones, and the sugars were eluted according to Spiro (1966) and quantified by the phenol sulfuric acid method (Dubois et al., 1956). For each sugar fraction, a standard curve was produced. To test the recovery, one set of standards on each sheet, containing 100/~g of each sugar, was left unstained and quantified in the same way as the samples. As an alternative, the amount of total hexoses present in the unhydrolyzed vitellogenin and vitellin was determined using the anthrone reaction (Roe, 1955) with mannose as a standard, and methyl pentoses were quantified by the Dische-Shettles cysteinvsulfuric acid reaction (Spiro, 1966) with fucose as a standard. Sialic acids were measured using the thiobarbituric acid assay after hydrolysis of 3 to 4 mg of protein with 0.5 ml of 0.1 N H2SO4 for 1 hr at 80°C (Spiro, 1966) with N-acetylneuraminic acid as a standard. For the quantitative analysis of hexosamines, 4-7 mg of protein were hydrolyzed in sealed tubes with 2 mi of 4 N HCI for 6 hr in a boiling water bath, the hexosamines were separated from the neutral sugars on cation exchange resins, and quantified by the Elson-Morgan reaction (Boas, 1953) with glucosamine hydrochloride as a standard.

Determination of lipids

Analyses were carried out in a Beckman Spinco model E analytical ultracentrifuge equipped with a monochromator and a photoelectric scanner. Sedimentation velocity was carried out at 52,000 rev/min in 12 mm double sector ceils made of Epon and KeI-F (according to the pH of the solution). The An-D and An-F rotors were kept at 20°C during the run. The moving boundaries were recorded at 280 rim. The molecular weight was determined by sedimentation equilibrium (Schachman and Edelstein, 1973) at 4000-12,000rev/min using an An-F and An-G rotor and charcoal double-sector cells filled with 0.1 ml solution (0.50.D.) and 0.12 ml buffer. No bottom oil was used. Data were analyzed with an Apple II computer.

Lipids were extracted from aqueous solutions of oocyte homogenates and from lyophilized samples of the purified vitellogenin and vitellin by the method of Reed et al. (1960). The extracts were dried under reduced pressure at 37°C. The dry residue was redissolved in chloroform-methanol (I:I, v/v), and aliquots of this solution were taken for the determination of cholesterol (Ott et al., 1982) and for qualitative and quantitative analysis of phospholipids. Total lipid phosphorous was quantified by the method of Rouser et al. (1970). Qualitative analysis was performed using the two-dimensional thin layer chromatography technique of Broekhuyse (1969), and the separated lipids were identified after staining successively with iodine vapor, ninhydrin (1% in acetone), and molybdenum blue (Dittmer and Lester, 1964). For quantification of individual phospholipids, the lipids were visualized using iodine vapor, scraped from the chromatography plate, and transferred into test tubes for phosphorous determination according to Rouser et al. (1970). As a control, silica-gel from a lipid free part of the chromatography plate was used.

Density gradient centrifugation

Amino acid analysis

Density gradient centrifugation on 5-30% (w/v) linear sucrose gradients was performed according to Martin and Ames (1961). The gradients were prepared with a Beckman syringe gradient mixer. The protein samples were extensively dialyzed against the same buffer which had been used for preparing the sucrose gradients. Centrifugation was carried out at 40,000 rev/min at 4°C for 15 hr with MSE 6 x 14 ml titanium swinging bucket rotor in a MSE superspeed 65 preparative ultracentrifuge. After completion of a run the tubes were emptied with a peristaltic pump from the bottom and fractions of 0.25 ml were collected for analysis with rocket immunoelectrophoresis.

Purified protein samples were dialyzed against distilled water, lyophilized, and delipidated (Kunkel and Pan, 1976). After hydrolysis in 6 N HCI containing 0.1% phenol and 0.05% 2-mercaptoethanol for 22 hr at 150°C, analyses were carried out in a Liquimat III amino acid analyzer (Kontron) with a Durrum DC-4A column and the Pierce Picobuffer system II.

Analytical ultracentrifugation

Determination of carbohydrates For the determination of carbohydrates, affinity-purified vitellogenin and vitellin preparations were extensively dialyzed against distilled water, lyophilized, and weighed on a Cahn electrobalance. Neutral sugars were obtained after

Partial specific volumes, calculation of molecular weight and frictional ratio fifo Partial specific volumes were calculated from amino acid composition data (Schachman, 1957) assuming specific volumes of 0.640 and 1.093 respectively for the carbohydrate and lipid components (Kunkel and Pan, 1976). Molecular weights and frictional ratios were calculated from specific volumes, sedimentation coefficients, Stokes radii and the density of the medium according to Siegel and Monty (1966).

356

H. IMBODENet al. RESULTS

Identification and molecular weight determination by P/IGE of vitellogenin and vitellin in Nauphoeta cinerea Since vitellogenins and vitellins of cockroaches do not appear as sharp bands in homogenous native PAGE (Engelmann et al., 1976; Harnish and White, 1982; Buschor and Lanzrein, 1983) we used gradient gels in tubes together with immunodiffusion tests for identifying vitellogenin and vitellin in Nauphoeta cinerea. Figure la shows the presence in both female hemolymph and oocyte homogenate of three closely spaced protein bands which are absent in the male hemolymph. In female hemolymph an additional female specific broad band is seen in the upper region of the gel, where three additional bands are also seen in the overloaded gel of oocyte homogenate. Densitometric scans of gel 3, obtained after staining with either Coomassie Blue or Sudan black, showed three peaks with both dyes, indicating that all three vitellins are lipoproteins (data not shown). We consider these female specific proteins to be vitellogenins and vitellins because the former accumulate in ovariectomized females, and the latter accumulate in the oocytes. V3 is not identical with the male and female band of similar migration behavior because oocyte homogenate gives no precipitation when tested with an anti-male antibody in Ouchterlony immunodiffusion tests (Bfihlmann, 1974). In addition, incubation of samples with antibody against male hemolymph prior to PAGE did not alter the appearance of the various vitellogenins and vitellins, whereas incubation with a vitellogenin specific antibody led to the disappearance of all vitellogenins and vitellins (data not shown). They were not seen in decapitated females but could all be induced by application of juvenile hormone III (data not shown). The Ouchterlony immunodiffusion test (Fig. lb) shows the presence of a very sharp and a very weak precipitation line in both female hemolymph (2) and oocyte homogenate (3), whereas no precipitate is seen with male hemolymph (1). This demonstrates female specificity and indicates that vitellogenin and vitellin are composed of the same two components. In vitellin the minor component is relatively more abundant. The molecular weight of the vitellins and vitel-

logeuins was determined using either native gradient PAGE in tube gels (Fig. la, c; Table 2) or native PAGE in slab gels of various concentrations of acrylamide (original data not shown but summarized in Table 2). The molecular weights are in the range of 300,000 and 600,000, the vitellogenins being of a slightly higher molecular weight than the vitellins. In slab gels of various concentrations of acrylamide only two closely migrating vitellogenins of approx. 300,000 could be distinguished together with the band of a higher molecular weight (Table 2).

Comparison with three other species (Leucophaea maderae, Hyalophora cecropia, and Manduca sexta) In order to verify whether our method produces reliable results we directly compared in native gradient PAGE in tubes male and female hemolymph as well as oocyte homogenates from Nauphoeta cinerea with those of Leucophaea maderae, a closely related cockroach species for which mol. wts of 270,000, 535,000, 790,000 and 900,000 for vitellin have been reported (data obtained by PAGE, Engelmann et al., 1976); we also included oocyte homogenates from the lepidopteran species Hyalophora cecropia for which a molecular weight for vitellin of 518,000 (sedimentation equilibrium method, Pan and Wallace, 1974) or 450,000 (gradient PAGE, Harnish and White, 1982) has been reported, as well as Manduca sexta, for which three vitellins have been reported (PAGE, Imboden and Law, 1983). The gels in Fig. 2a show that Leucophaea oocyte homogenate (gel 3) contains several viteUin bands (Mr 270,000-350,000) with a similar electrophoretic behavior to those of Nauphoeta (gel 6), and that similar to the situation in Nauphoeta the Leucophaea vitellogenin (gel 2) appears to be slightly larger (Mr 310,000-360,000) than the vitellin (gel 3). In comparison, the gels of the oocyte homogenates of the lepidopteran species Hyalophora (gel 8) and Manduca (gel 9) show that their vitellins have a higher molecular weight than those of the cockroaches. Calculations of the molecular weight reveal a value of 520,000 for Hyalophora and 510,000, 520,000 and 545,000 for Manduca. We also made a comparison between Nauphoeta and Leucophaea in an Ouchterlony immunodiffusion test (Fig. 2b), which showed partial cross-reactivity of the Leucophaea female hemolymph and oocyte homoge-

Table 2. Molecular weights of the vitellogenins (Vgs) and vitellins (Vs) determined using two PAGE methods Molecular weight ( x 103) Method of determination

Vitellogenin

Native gradient PAGE in tube gels*

303 (Vg3) 314(Vg2) 329 (Vgl) 590-628 ("hVg")

PAGE in slab gels of various concentrations of acrylamidet

299~. 324./ (Vgs) 578 ("hVg')

Vitellin 274 (V3) 293 (V2) 308 (V1) 550 ") 603 ~ ("hVs') 627..I ND

*Means of four determinations for the vitellogenins (SEM <2%), of 15 determinations for the vitellins (SEM <0.6%), and of three determinations of the "hVs" (SEM <0.6%). tAt least two determinations were made with each acrylamide concentration. ND = not determined.

357

N a t i v e v i t e l l o g e n i n a n d vitellin o f N. cinerea

nate with the antibody to female-specific proteins of

Nauphoeta, but the spur between (3) and (6) dearly indicates antigenic differences between the vitellins of the two species.

Molecular weight and S2o.wdetermination by the analytical ultracentrifugation, fiictionai ratio, role of pH and ionic strength Since it is known that vitellin displays pH(Brookes and Dejmal, 1968) and ionic strength(Dejmal and Brookes, 1968, 1972) dependent aggregation behavior and since our PAGE systems were high pH techniques, we also utilized analytical ultracentrifugation for molecular weight and s20.w deterruination of vitellin and tested the effect of pH and ionic strength (Table 3). The data obtained show that a low ionic strength Tris buffer at pH 8.25 (PAGE buffer) favors formation of an approx. 244,000-dalton unit with an s20,w value of 9.4 S. Hereafter we refer to this smallest vitellin detectable by the different methods as a monomer. The molecular weight of the vitellin found at pH 7.5 in low and high NaCI concentration (approx. 490,000) would thus represent a dimer. A low pH apparently favors formation of aggregates of different size. The value observed in the high pH glycine buffer indicates that in contrast to the PAGE buffer a portion of the vitellin is present as a dimer. The molecular weight values calculated from hydrodynamic parameters are somewhat different from those determined by the sedimentation equilibrium method. Calculation of the frictional ratios gave values between 1.67 and 1.45, the highest value being that of the monomer. This demonstrates that the vitellin monomer and dimer do not have a globular shape.

The effect of Triton X-IO0 on the vitellin as analyzed by sucrose density gradient centrifugation Having demonstrated that the vitellin can occur in different forms depending on analysis conditions we examined the effect of Triton X-100 on vitellin in a buffer system of 0.02 M sodium phosphate, 0.4 M

NaCI, pH 7.5. In this buffer system a s20,w value of 15 S has been determined using the analytical ultracentrifuge (Table 3). The curves in Fig. 3 show that the addition of Triton X-100 alters the sedimentation velocity and from a comparison with catalase and undissociated vitellin as standards an apparent s20,w value of 8.4 S for the Triton X-100 treated vitellin was calculated. This indicates that Triton X-100 induces disassembly of the vitellin dimers into monomers.

Determination of the Stokes' radius by gel permeation chromatography In order to characterize further the vitellogenin and vitellin we used gel permeation chromatography to estimate the Stokes' radius. Female hemolymph or oocyte homogenate was applied and eluted fractions were tested by immunodiffusion and/or rocketimmunoelectrophoresis for the presence of vitellogenin and vitellin. Both vitellogenin and vitellin eluted as a single sharp peak between thyroglobulin (Stokes' radius 8.5 nm) and ferritin (Stokes' radius 6.1 nm; curve for vitellin shown in Fig. 4a) and a comparison of their elution volume with that of the standard proteins revealed a Stokes' radius of 7.52+0.02nm ( N = 4 ) for vitellogenin and of 7.61 +0.04nm (N =6) for vitellin (Table 3) in 0.02M sodium phosphate, 0.4M NaCI, pH7.5. Under conditions in which vitellin is present as a monomer (PAGE buffer or 0.4 M NaCI, pH 8.8), we found a Stokes' radius for vitellin and vitellogenin of 7.5 nm (Table 3 and data not shown). The careful analysis of the fractions by immunodiffusion (Fig. 4B) showed that a second minor vitellin can be seen in fractions 51-61. Determination of the Stokes' radius for this minor component (peak in fraction 58 according to rocket immunoelectrophoresis, data not shown) gave a value of 4.8 nm.

Carbohydrate content and composition Since vitellogenin and vitellin both stained with the periodic acid-Schiff's reagent after electrophoresis in polyacrylamide gels and precipitated with Con-

Table 3. h0.w, molecular weights (analytical ultracentrifugation), Stokes' radius and frictional ratio of vitellin in various buffers Molecular weight g/ml

s20.w

Sedimentation equilibrium

Calculated from a, s20,w, v*

0.02 M sodium phosphate 0.4 M NaCI, 0.02% NaN 3 pH 7.5

1.020

15.0

491,000

493,000

0.02 M sodium phosphate 0.08 M NaC1, 0.02% NaN 3 pH 7.5

1.003

15.2

485,000

499,000

PAGE buffer 0.015 M "Iris + 0.11 M glycine pH 8.25

1.002

9.4

244,000

309,000

0.1 M glycine/HCI 0.4 M NaCI, 0.02% NaN 3 pH 3.6

1.015

0.1 M glycine/NaOH 0.4 M NaCI, 0.02% NaN 3 pH 9.3

1.015

15.8 + 30

9.4

Stokes' radius (a) (nm)

fifo

7.61_+0.04 1.45 (N -- 6)

7.5 (N = I)'["

1.67

811,000 + 1,896,000

325,000

*For calculation of the molecular weights and the frictional ratio f/f0 the method of Siegel and Monty (1966) was applied, using a partial specific volume (v0) of 0.7417 cm~/g (see Results). t i n a buffer of 0.4 M NaCI, pH 8.8, where vitellin is present as a monomer we also found a Stokes' radius of 7.5.

H. IMBODENet al.

358

the unhydrolyzed protein fractions by the anthrone reaction, using mannose as a standard, yielded the same values as determined by the phenol sulfuric acid method. Neither methyl pentoses nor sialic acid could be detected, whereas vitellogenin and vitellin both contained hexosamines. The difference in the mannose content of vitellogenin and vitellin was not significant (significance level P > 0.05) and the ratio of mannose to hexosamines was 3.5:1 for both proteins.

50-

40E 30g 2O8

/ !?[i !

A \

Lipid composition

\

n.

101! 0 ....... ,'~/ 0

8

I 16

24

Froc)Jonnumber

32

40

Fig. 3. Effect of Triton X-100 on the vitellin as analyzed by sucrose density ccntrifugation. 300/zl of a freshly prepared oocyte (days 10-11) homogenate (protein concentration 1.80.D.) was analyzed in 0.02 M sodium phosphate, 0.4 M NaC1, 0.02% NaN 3, pH 7.5 (open triangles) or in the same buffer containing 1% Triton X-100 (solid triangles). The relative concentration of vitellin (ram rocket height), as measured by rocket immunoelcctrophoresis, is given for each fraction. canavalin A in agarose gels (data not shown), they can be considered glycoproteins. Qualitative analysis of the neutral sugars of vitellogenin and vitellin by paper chromatography and staining with aniline phthalate revealed the presence of only mannose in both proteins. The quantitative carbohydrate analyses are summarized in Table 4. Mannose was the only hexose found. Determination of total hexoses in Table 4. Carbohydrate composition of vitellogenin and viteUin Component

Vitellogenin

Mannose by PC* Mannose by aothronet" Methyl pentoses Sialic acids Hexosamines

5.9 + 0.3 5.9 _+ 0.3 1.7 _+ 0.2

(3) (8) (5) (3) (4)

Vitellin ND 5.5 + 0.2 (6) (2) (3) 1.6 _+ 0.3 (4)

*Determined using quantitative paper chromatography and the phenol--sulfuricacid method; ND = not determined. tDetermined using the anthrone reaction. Data arc expressed as a percentage of total weight and given as means+ SEM of (N) determinations.

Vitellogenin and vitellin both stained with Sudan black B after electrophoresis in polyacrylamide gels (data not shown) indicating their lipoprotein nature. In preliminary lipid analyses using chloroformmethanol (2:1, v/v) extraction according to Bligh and Dyer (1959), values for total lipid of 4.8 and 7.4% were found for purified vitellin and vitellogenin respectively. We observed that oocyte homogenates contained phospholipids (66.8 #g/rag protein) and very little cholesterol (2.3 #g/rag protein). The phospholipid composition of the purified protein preparations is summarized in Table 5. Both vitellogenin and vitellin contained the same phospholipids, which differed, however, in their quantitative distribution in the two proteins. Vitellogenin contained significantly more phosphatidylcholine and lysophosphatidylcholine than vitellin. The proportion of phosphatidylethanolamine on the total phospholipid content was correspondingly increased in the vitellin. N o sphingomyelin was detectable.

Amino acid composition Amino acid analysis revealed no striking differences between vitellogenin and vitellin (Table 6). Both proteins were rich in aspartate/asparagine and glutamate/glutamine as well as in serine and leucine, whereas the content of methionine was low in both.

Partial specific volumes The apparent specific volumes of the pcptide components of vitellogenin and vitellin were calculated from the amino acid composition and gave values of 0.7302cm3/g for vitellogenin and 0.7279cm3/g for vitellin. The partial specific volumes of the entire lipoglycoproteins are 0.7477cm3/g for vitellogenin and 0.7417 cm3/g for vitellin. DISCUSSION

Multiple vitellogenins and vitellins In immunodiffusion tests (Fig. l b) an intensive sharp and a weak broad precipitation line c o m m o n

Table 5. Content and compositionof phospholipid of viteUogeninand vitellin Vitellogenin Vitenin Total phospholipid* 6.7__0.8% 77.9+ 8.8#g/mg 5.5 +0.5% 62.4+ 5.6#g/mg NS Phosphatidylethanolaminet 14.8 + 0.4 11.5 23.0 + 1.8 14.4 :~ Phosphatidylserine 13.0 + 1.0 10.1 16.9 + 1.4 10.5 NS Phosphatidylcholine 59.8 _+2.1 46.6 53.5 _+1.8 33.4 § Lysophosphatidylcholine 12.4+ 2.5 9.7 6.6 + 1.2 4. I :~ *Total phospholipid is expressed as a percentage of the total weight and as/~g/mg protein. tlndividual phospholipids are expressed as percentages of total phospholipid. Values in pg/mg were calculatedfrom the total phospholipidcontent of 77.9gg/mg for viteUogeninand 62.4#g/rag for vitellin. Difference betweenvitellogeninand vitellin significant:~P < 0.025; §P < 0.05; NS = not significant.Mean values_+SEM from four determinations.

a

hVs b

V1 V2 V3

C

iiJ~!iiiii~ii~Jiiiiiiii¸ ii!

d

2

3

4

3+5

(A)

5

(c) 10 6 _

5x10

5

..~j,-Vg

2

(D

1o 5

I

I

2O

4O

•

Distance travelled

I

I

60

80

(ram)

Fig. 1. Identification and molecular weight estimation of vitellogenin and vitellin of Nauphoeta cinerea. (.4,) Native gradient PAGE in tubes of hemolymph of 1I-day-old males (1), hemolymph of 1l-day-old females (2), ooeyte homogenate from 11-day-old females (3) and (4) the latter overloaded, as well as molecular weight standard proteins (5), namely thyroglobulin 669,000 (a), ferritin 440,000 (b), catalase 232,000 (c) and lactate dehydrogenase 140,000 (d). VI, V2, V3 denote vitellins; hVs = high molecular weight vitellins. Bars in lane 2 denote vitellogenins. (B) Ouchterlony immunodiffusion of male hemolymph (1), female hemolymph (2) and oocyte homogenate (3) with a female-specific antiserum in the center hole. (C) Semilogarithmic plot of the molecular weights versus the migration distance (Lorentz, 1976). Arrows point to the positions of the viteUogenins (Vg) and vitellins (V).

359

1

2

3

4

5

6

6+7

8

9

7

(A)

Fig. 2. Comparison with other species. (A) Native gradient PAGE in tubes of Leucophaea maderae male hemolymph (1), female hemolymph (2) and mature oocytes (3); of Nauphoeta cinerea male hemolymph (4), female hemolymph (5) and mature oocytes (6); of Hyalophora cecropia mature oocytes (8) and of Manduca sexta mature oocytes (9) together with molecular weight markers (7). (B) Ouchterlony immunodiffusion of Leucophaea rnaderae male hemolymph (1), female hemolymph (2) and mature oocytes (3) and of Nauphoeta cinerea male hemolymph (4), female hemolymph (5) and mature oocytes (6). The center hole was filled with an antiserum against Nauphoeta "female-specific proteins.

360

(A) bcd

O.B

E

0.6

c

o ~d ~

0.4

~ <

0.2

g

.Q

20

40

60

80

I00

FFoction number

Fig. 4. (A) Gel permeation chromatography of oocytc homogenate of Nauphoeta cinerea. (a) Thyroglobulin, (b) ferritin, (c) catalase, (d) aldolase. (B) Analysis of gel permeation chromatography fractions nos 30-66 of oocyte homogenatc by immunodiffusionwhereby slits were filled with either antibody against male hemolymph or antibody against female specific proteins (anti vit).

361

Native vitellogenin and viteUin of N. cinerea Table 6. Amino acid composition of vitellogenin and vitellin Vitellogenin Amino acid

Asp Thr Scr Glu Pro Gly Ala Val Met lte Leu Tyr Phe Lys His Arg Cys Trp Total

Vitellin

mol/mol tool % mol/mol mol % 342.8 125.8 181.9 242.4 128.1 89.0 131.0 159.2 45.6 117.1 193.7 99.1 108.6 164.0 84.4 146.9 ND ND

14.5 5.3 7.7 10.3 5.4 3.8 5.6 6.7 1.9 5.0 8.2 4.2 4.6 7.0 3.6 6.2

335.5 112.3 195.9 240.0 119.3 73.3 114.8 153.4 37.3 109.5 176.0 88.7 97.1 150.8 75.3 143.3 ND ND

2340.6

100.0

2239.5

15.1 5.1 8.8 10.8 5.4 3.3 5.2 6.9 1.7 4.9 7.9 4.0 4.4 6.8 3.4 6.4

100.0

The results represent uncorrected data (moles amino acid per mole peptide and mole per cent) and are based on a molecular weight of 270,000 for vitcllogenin and of 255,000 for vitellin (means of the three vitellogenins and viteUins respectively, after subtracting the carbohydrate and pbospholipid moiety). ND ~ not determined. Values represent means of three determinations.

to both vitellogenin and vitellin were seen, indicating that both consist of the same major and minor components. The data obtained in gel permeation chromatography (Fig. 4) suggest that the minor component is a much smaller molecule (Stoke's radius of 4.8 nm) than the major component (Stokes' radius of approx. 7.5 nm for the monomeric and dimeric form of vitellin). We do not know whether the minor component is an immunologically distinct vitellin but heterogeneity of vitellogenins and/or vitellins with respect to immunological properties has been observed also in other species of cockroaches (Barth and Bell, 1970, Bell, 1970; Storella et al., 1985). Using native gradient PAGE in tubes we found three closely spaced bands and an additional weak and broad band of vitellogenin in female hemolymph and three closely spaced vitellins in oocytes of Nauphoeta cinerea (Fig. 1). This method is very efficient as seen from the resolution of five bands for the molecular weight marker catalase and the comparison with earlier results (Buschor and Lanzrein, 1983). Multiple vitellogenins and/or vitellins with respect to their electrophoretic behavior have also been observed in Aedes aegypti (Hagedorn and Judson, 1972), Acheta domesticus (Bradley and Edwards, 1978), Rhynchosciara americana (De Bianchi et aL, 1982), Manduca sexta (Imboden an.d Law, 1983 and Fig. 4), Rhodnius prolixus (Masuda and Oliveira, 1985) and the cockroaches Leucophaea maderae (Engelmann et al., 1976) and Periplaneta americana (Storella et aL, 1985).

Molecular weight determination using different methods, role of pH, ionic strength and effect of Triton X- IO0 Molecular weight determinations under various conditions by analytical ultracentrifugation (Table 3) LB. 17/2--G

363

reveals that vitellin appears as a 244,000 dalton protein in PAGE buffer (pH 8.5) but as an approx. 490,000-dalton unit in a buffer of pH 7.5. From this we conclude that the native vitellin is a dimer which disassembles in a high pH-environment. In glycerol density gradient ultracentrifugation in a phosphate buffer of pH 6.5 vitellogenin and vitellin were both seen as an approx. 15 S component (K6nig, unpublished observation), indicating that the native vitellogenin is also a dimer. The s20.w values of the monomer and dimer are 9.4 and 15 S respectively, the Stokes' radius is approx. 7.5 nm for both and the frictional ratio is 1.67 and 1.45 respectively. These data suggest that the monomer and dimer are elongated molecules and that their longest axis is almost the same. By the use of scanning transmission electron microscopy similar observations have been made with vitellin of Blattella germanica (J. G. Kunkel, personal communication). In sucrose density gradient centrifugation the vitellin with a s20,w value of 15 S according to Table 2 disintegrates to a vitellin with a value of 8.4 S when treated with Triton X-100 (Fig. 3). In accordance with observations made for other proteins (Simons et al., 1978) this strongly suggests that hydrophobic interactions among the vitellin monomers are responsible for the formation of vitellin oligomers (see also Table 3). In Leucophaea maderae, oocyte homogenates contain a 14 S component with a Mr of 559,000 and a 28 S component with a Mr of 1,590,000 (Dejmal and Brookes, 1972); during development of the oocytes the relative proportions of the two components change, the 28 S becoming predominant towards the end of the oocyte maturation cycle (Brookes and Dejmal, 1968; Koeppe and Ofengand, 1976). Under comparable conditions with respect to oocyte maturation stage and pH, Leucophaea maderae vitellin consists of 82% of the 28S and only 18% of the 14S component (Dejmal and Brookes, 1972) whereas Nauphoeta cinerea vitellin exclusively contains a 15 S component (Table 3). In Blattella germanica formation of a 33 S component from 18 S forms is seen after chorion formation (J. G. Kunkel, personal communication). In a preliminary analysis of Nauphoeta cinerea egg cases immediately after ovulation using glycerol density gradient ultracentrifugation we observed that approximately half of the vitellin behaved as an approx. 30 S component, whereas none of this was seen in mature oocytes shortly before chorion formation (K6nig, unpublished observations). This suggests that after chorion formation a comparable aggregation of vitellin occurs also in this insect. Thus, the 15.8 and 30 S forms observed at pH 3.6 (Table 3) might correspond to aggregates which naturally appear at later stages of oocyte development. The specific volume of the Leucophaea vitellin as determined pycnometrically is 0.756cm3/g (Dejmal and Brookes, 1972), and that of Bianella germanica vitellin 0.755 cm3/g, as calculated from composition data (J. G. Kunkel, personal communication); these values are similar to that of 0.7417 found in Nauphoeta cinerea. The direct comparison of Nauphoeta vitellin in native gradient PAGE with vitellins of another closely related cockroach species (Leucophaea) and of two lepidopterous species (Manduca and Hya-

364

H. IMBODENet al.

lophora) shows that the cockroach vitellins have lower molecular weights than the lepidopterous ones under these conditions (Fig. 2A). Published molecular weights for the Leucophaea vitellin determined by PAGE were 270,000 and 525,000 (Engelmann et al., 1976). With our native gradient PAGE system we found that the vitellins from Nauphoeta and Leucophaea exhibit similar electrophoretic behavior and molecular weights of approx. 300,000. For Manduca, Mundall and Law (1979) described one vitellin with a molecular weight of 260,000 using gel permeation chromatography, whereas Imboden and Law (1983) detected three vitellins in PAGE. We confirmed the presence of three vitellins in Manduca and showed that they have molecular weights of 510,000, 520,000 and 545,000, similarly to the vitellin of Hyalophora (520,000). The similarity in structure of the vitellins of these lepidopterous species is further supported by high performance liquid chromatography studies that indicated the same retention time for Manduca and Hyalophora vitellin (data not shown). In addition, our result for the Hyalophora vitellin is in good agreement with the molecular weight published by Pan and Wallace (1974). Obviously the lepidopteran vitellins do not disassemble in the PAGE buffer, as is the case for the cockroach vitellins. Carbohydrate, lipid and amino acid composition The carbohydrate moiety of the vitellogenins and vitellins of Nauphoeta is entirely made up of mannose and N-acetyl hexosamine (Table 4) and no significant differences between vitellogenin and vitellin were observed, as was the case in all other species where comparative analyses have been made (Kunkel and Pan, 1976; Hagedorn and Kunkel, 1979; Chino et al., 1977; Mundall and Law, 1979). Total lipid was found to be around 7.4% for vitellogenin and 4.8% for vitellin in preliminary analyses using chloroform-methanol extraction in Nauphoeta cinerea, but these values may not be very precise because of the method applied and because it is not known whether all lipid is left intact during the purification procedure. In the cockroach Leucophaea maderae a value of 6.9% for vitellin is published (Dejmal and Brookes, 1972) and in Blattella germanica values of 7.6% for vitellin (Oie et al., 1975; J. G. Kunkel, personal communication) and of 15.7% for vitellogenin (Kunkel and Pan, 1976). The latter value, however, was obtained with vitellogenin from ovariectomized females which have an altered lipid metabolism. Analyses of the phospholipid moiety in Nauphoeta (Table 5) revealed that vitellin contains significantly less phosphatidylcholine and lysophosphatidylcholine than vitellogenin. Thus, these phospholipids seem to be released within the oocyte in a specific degradation process since the absolute quantities of the other two phospholipids detected do not differ between vitellogenin and vitellin. The amino acid composition of vitellogenin and vitellin in Nauphoeta (Table 6) is very similar to that in other insects (Hagedorn and Kunkel, 1979) and no significant difference between vitellogenin and vitellin could be seen, as was the case in other insects (see Hagedorn and Kunkel, 1979; Mundall and Law, 1979; Chinzei et al., 1981). Amino acid analyses of

viteUogenins and vitellins may prove useful for investigating evolutionary aspects of these proteins. In conclusion we present evidence that the native vitellogenin and vitellin of Nauphoeta einerea consist of a major component, namely a dimeric molecule with an elongated shape, and of a minor and much smaller component. Under high pH conditions and under the effect of Triton X-100 the dimers disassemble into monomers, the latter being heterogenous with respect to their electrophoretic mobility. No major differences in the physico-chemical properties of the native vitellogenin and vitellin have been detected but work in progress suggests some differences with respect to their peptide composition. Acknowledgements--We wish to thank Mrs A. Tschan for rearing the cockroaches and Professor J. G. Kunkel for helpful comments on the manuscript and for showing results of unpublished work. Financial support by the Swiss National Science Foundation (grants 3.714-80 and 3.291-0.82 to B. Lanzrein and 3.3584).82 to P. Ott) is gratefully acknowledged. REFERENCES

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