Comparative structural studies of vitellogenin and vitellin of Rhynchosciara americana

pp. 895--900, 1985

'~ATIVE STRUCTURAL S N A N D VITELLIN OF /~ AMERICANA A. G. DE BIANCHI and S. D. PERE1] imica, Instituto de Quimica, Universidad~ ~P: 01498--Sgo Paulo, Brasil (Tel: 011-21

OF 9SCIARA

, C. P. 20780,

(Received 3 July 1984) )genin and v Abstract--1. A procedure for labelling R. americana vitello[ developed. 2. The four types of vitellogenin and vitellin subunits were wer, labelled b fat bodies. a 3. A bidimensional electrophoretic analysis shows that vitellogenin vii 3totems. isoelectric points and apparent mol. wts in the two proteir )resent in vitellog 4. The number of the different polypeptide types presenl 5. The obtained results imply that differences between app)arent mol. tifferential cc R. americana vitellogenin and vitellin may be due to a diJ

INTRODUCTION most of the insects that have been studied, the incipal proteins of the eggs, the vitellins, are rived from the vitellogenins synthesized by the fat derived bodies. Vitellogenins and vitellins from the same ;ect species are immunologically identical and the insect apoproteins frequently have identical structures (Engelmann, 1979; Hagedorn and Kunkel, 1979). )wever, some cases of differences between vitelHowever log.genin and vitellin apoproteins have been reported ,oeppe and Offengand, 1976). The vitellins of insect (Koepl ma'xy also differ from vitellogenins in the amount of associated lipids, even thoutgh the apoprotelns are identical (Chinzei et al., 1981). Vitellogenin and vitellin ofpf R. americana are lipoglycophosphoproteins with hifigh mol. wts (Bianchi et al., 1982; Pereira and Bianchi, 1983). These two proteins exhibit differences in their mol. wts and ad Bianchi, 1983). Both isoelectric points (Pereira and vitellogenin and vitellin are composed of four types of polypeptides (V1, V2, V4 and and V5) having the same mol. wts in the two proteins Is (Pereira and Bianchi, 1983). In this paper, we report a comparative analysis of belling of R. americana these proteins based on labellin 32p_orthophosphate" vitellogenin and vitellin by 32p-orth( rtho [Jx aaa~.zl.,~z

MATERIALS AND) METHODS Rhynchosciara americana wass reared in the laboratory )varies ha haemolymphandfat according to Lara et al. (1965). Ovaries ed by Bianchi et al. (1982). bodies were obtained as described Yolk granules were obtained ass described by Pereira and Bianchi (1983). Vitellin was extratcted from the yolk granules by shaking with 0.025 M Tris~, 0.192M glycine, 5raM EDTA, pH 8.3, for 2 hr at 4°C.7. The resulting suspension w 10min at 4°C and the was centrifuged at 16,300g for supernatant was used as vitellint preparation. . . . Incorporation of 32p-orthophosphate in vi Recently emerged female flies were 32p-orthophosphate diluted in sugar solu r- la D g n : a n

rthophosphate was ~sphate only by the anits have identical in is identical. tric points between ; in these proteins.

this time, the haemolymph and le: left for 4 hr were dissected. The was {tcollected .,ott~,~ t~,~ aaz l ~ t taa~. U V Q X lies ~O haeme haemolymph was dilluted with sample buffer for electrophores Bianchi et al., 1982). The horesis in the presence of SDS (Blanch ~horesis in the presence of ovanes ovarie were prepared for electrophoresi., vitellin (see . SDS (Bianchi et al., 1982) or used to obtain above). 7oration of 32p-orthophosphate in vitro v lncorp tbated for 2 hr at 25°C on Fat bodies and ovaries were incubated Fat siliconized coverslip in 20 #1 of a ph) ,siological solution a silic~ (Terra et al., 1976) in the presence of 40#Ci 32p.or :P-orthophosphate. After incubation, tthe ovaries and fat bodies were submitted to immunoprecip)Ration (see below) or solubilized in sample buffer for "or electrophoresis electr (Bianchi et al., 1982). Electrophoretic methods )horesis in the presence of Slab polyacrylamide gel electrophoresi et aL (1982). SDS was carried out as described by Bianchi Bi :rformed as described Bidimensional electrophoresis was perfe by O'Farrel et al. (1977). The materials to be submitted to this procedure ( 3 2 p labelled ovaries, 32p 32 labelled vitellin preparations and 32p labelled haemolyml:,h) were solubilized in "lysis buffer" (9.5 M urea, 2% (v/v) Nonidet P-40, 2% (v/v) "pharmalyte 3-10" and 5% (v/v) fl-mercaptoethanol) fl and focused in a polyacrylamide column (130 x 2.5 mm)for 4 hr at 400 V. The second dimension sep9aration was run on a 10% polyacrylamide-SDS slab. After the run, the slabs and submitwere stained with Coomassie blue R,, destained, des1 1983). ted to autoradiography (Pereira and Biancbi, Bi; Limited proteolysis of vitellin and vitelloj,genin was carried out as Limited proteolysis by papain wa Haemolymph, ovaries described by Cleveland et al. (1977). Ha and vitellin preparations, labelled with 32P-orthophosphate, :rylamide gel slab electrowere submitted to 10% polyacrylamid¢ were stained using a phoresis. After the run, the slabs wet solution of 0.1% (w/v) Coomassie Blue R I in 50% methanol containing 10% acetic acid. containing After destaining in a solution of 5% methanol m 10% acetic acid, the regions of the slab sl containing the tides (V I + V2, V4 and Vs) i min in a solution contain1% SDS and 5 mM EDTA,

A. G. DE BIANCH! and S. D. PEREIR~ slots of a 15°,o iquot of 10pl of the 3~o (v/v) glycerol was dditional 10#1 of the glycerol and defined was carried out at tracking dye reached was disconnected for peptides to occur. The ted and the resulting y.

itation ~2p-orthophosphate-labelled ovaries and fat bodies were mogenized in 200#1 of 25mM Tris, 192raM glycine ffer, pH 8.3, containing 1.5~o (w/v) sodium deoxycholate, (v/v) Triton X-100, l mM phenylmethylsulphonyl aride and 0.1~ (w/v) NaN3 and centrifuged at 20,000g 10rain at 4°C. The supernatants were submitted to munoprecipitation using the antiserum described by :eira and Bianchi (1983) and the method described by ssler (1975); the washing of the final pellet was carried out described by Marinotti and Bianchi (1983). The immuprecipitated materials were solubilized in sample buffer electrophoresis in the presence of SDS (Bianchi et al., ~2). lative stoichiometry of vitellogenin and vitellin subunits in order to determine the relative proportions of constitit polypeptides (V~, V2, V4 and V s) present in vitellogenin t vitellin, the radioactivity of each polypeptide was e m ployed to calculate the ratios dpm in V l + V2/dpm in V4, dpm rn in V I + V2/dpm in V 5 and dpm in V4/dpm in V 5. For this purpose, five aliquots (1, 2, 5, 10 and 20 ktl) of 3 independent 32p-orthophosphate labelled samples of haemol'lymph, vitellin preparations (see above) and ovaries were >mitted to 10~o polyacrylamide-SDS slab electrophoresis. submitted Thee polypeptides (V~ + V2), V4 and V5 were excised from the

a .....

b

e

Ii

m

i

gels and the radio and Bianchi (19 molymph, vitellin by least squares 1 where dpm repre and Vol is the cc applied to the gel the relations a w logenin and vitell variance analysis (Battler's test of

ined as described by Pereira 3 material analysed (haend ovary) the data were fit of the type: ol+h rved radioactivity (in dpm) olume (in #1) of the aliquot ) were employed to estimate 2/av5 and av4/av~ for vitelin turn intercompared by a mptions were tested a priori fJohnson and Leone, 1964). TS

Label Labelling o f vit

vitellin

Vitellin of R. s a p h o s p h o p r o t e i n with Vit aa ppho hosphorus c ~°/o (Pereira and Bianchi, 1983), pointin )ossibility of labelling vitell( vitellogenin an( 2P - o r t h o p h o s p h a t e . The electrophoretic diographic analyses of haem, haemolymph p ned from adult females fed w with 0.1 m ( o p h o s p h a t e is displayed in Fig. la. T h a:s that the vitellogenin ssubunit u b u r polype t , V4 a n d V5 (Pereira a n d Bianchi, 1983) the only h a e m o l y m p h by 32P - o r t h o p h o s p h a t e . c o m p~onents he; In the th~ ovaries ( e other polypeptides are also labelled 1 in addition to vitellin subunits. Ovaries i n c u b a t e d in vitro with 2 0 # C i of 32p_01 P - o r t h o p h o s p h a t e do not show labelling la of the V~ a n d ~, V2 polypeptides (Fig. lc). Since the results were a m b i g u o u s as to whether V4 and V5 were labelled, ambi~ labelled ovary proteins were i m m u n o p r e c i p i t a t e d with anti-vitellin antiserum. Since the immu-

d

e

4bv ii!!

t

IVv:

.........

m

>hate. Recently hic analysis of R. americana proteins labelled by 32p-orthophosphate Fig. 1. Autoradiographic ere fed with 32P-orthophosphate and, after 4 hr the haemolymph (a) aand ovarian emerged female flies were :sed by auto(b) polypeptides were subjected to electrophoresis in the presence of SDS and analysed bodies of recently radiography. (c) Ovary incubate munoprecipitate of emerged female flies incubated /peptides. incubated fat bodies,

R. americana vitellogenin and viteilil )t labelled and the vitellinic subunits that none of the d when ovaries were ate. begins to be syn)f pupal life (Bianchi , 1983). The/n vitro ently emerged adult shows that all of the phorylated by this

Bidimensional and vitellin su~ ViteUogenin different isoeh 1983). Since t] differences in l polypeptides, phoretic analy~ was carried ou The results (i and apparent peptides are id

ic analysis of vitellogenin of R. americana have (Pereira and Bianchi, ,' might be traceable to : pHs of the constituent e bidimensional electrorain and vitellin subunits that the isoelectric points bf the vitellogenic polyose of the vitellinic sub-

I

?

i!~iliiii!i~iiiii

Fig. 2. Autoradiographic analy subjected to two-dimensional el autoradiogram A was positiom th~ nntc~rncllnerr~m I~

)rthophosphate and [lin preparation; (C) was positioned over lypeptides.

A. G. DE BIANCHIand S. D. PEREIRA

i

2

3

4

1 s

! i :e

L

i

8

'4 and V, ) obtained rg; 5,11 V, o f Vg: " Vt: 1 2 - V , c)l" VL

Fig. 3. Limited papain digestion of the vitellogenic (vitellinic) (vitellini polypept from haemolymph, ovary and vitellin preparation: t,7--V~ + V: of V~ 2 V~ + Vz of Vt ov; 4~V 4 of Vt ov; 6~V~ of Vt ov: 88-- V l + V2 of Vg vitellogenin: Vt ov ovary vitellin; Vt vitelli

its. It is important to note that the vitellinic lypeptides from both the vitellin preparation :tracted from yolk granules) and the ovary are (extracted ;ntical, suggesting that the vitellin preparation identical ~cedures (see Materials and Methods) do not introprocedures ce any detectable modification into the vitellinic duce polypeptides. ~ited proteolysis o f vitellogenin and ritellin subunits Limited Using 32p-orthophosphate labelled haemolymph, Usin ellin preparation and ovary, a comparison of the vitellin ellogenic and vitellinic subunits was made using vitell iod. The results (Fig. 3) the limited proteolysis method. show that vitellogenic andl vitellinic polypeptides ,'rns when hydrolysed by have identical cleavage patterns papain. The observed diffe,~rence between haemoage pattern observed in lymph polypeptide V 4 cleavage :ig. 3, lane 3 and lane 9) two different experiments (Fig. are due to different degrees of hydrolysis. ellogenin and vitellin s'ubRelative stoichiometry of vitellog~ units The observed difference bbetween the mol. wts of vitellogenin and vitellin (Peree i r a a n d Bianchi, 1983)is much too small to permit postulation of a m o n o m e r ~ l i m e r relationshiLp. Since the subunits of bility is a difference in the both are the same, one possibility mnits. To examine this relative ratio of these subu pproach was employed to possibility, a comparative app

netrv of vitellogenin and analy/se the rela :ellin subunits (see Materials and Methods). vitelli The Th, relations between the polypeg)tides V~ + V,, V~ vitellin, labelled using a n d Vs in vitellogenin and vitelli 32p-orthophosphate, are shown in Table 1. Statistical 32P-o comparison of all three relations (av~+,,2./av~: enin with those of a V I + V v21'av5 and av41'av~) of vitelloge ellin indicates that the relative q uantities of these vitelli polylc in both proteins. It ~olypeptide subunits are the same ir lations obtained for is i mLportant to note that the relati procedure ellin are not modified by the extraction exti vitellJ ~aration. The results used to obtain the vitellin prepare point to the conclusion that not only the subunit composition, but also the subunit ,,stoichiometries of mertcana are the same. vitellogenin and vitellin of R. ameri~

DISCUSSION americana are high Vitellogenin and vitellin of R. at )rotems (Bianchi et al., mol. wt lipoglycophosphoproteins two proteins 1982; Pereira and Bianchi, 1983). These T exhibit different mol. wts and isoelectric points (Pereira and Bianchi, 1983). vitellin is The phosphorus bound to R. americana an localized on serine residues of the th( protein (Pereira that the and Bianchi, 1983). Our results, demonstrate der are phosfour vitellogenic and vitellinic subunits su phorylated only by the fat bodies. Thus, unlike the

Table 1. Ratios between the polypeptides subunits V~+ V2, V4 and "v'~of R. "~mericana vitellogeninand vitellin labelled by 32P-orthophosphate a~ M aterial*

Vitellogenin itellin (ovary) Vitellin itellin preparation Vitellin kJl,~tpvi~l¢ aan d M e t h n d q *See Materials tAngular coetfic (/d) + b: wh **Mean_+SEM f

11 12

av I ~ V2 "¢lv4 ~"

t/VI ~ \'2 ,'av5

3.63_+0.25+ 3.33-+0.13 3.06 -+0.14

5.20+0.19 4.98 + 0.31 4.78 -+0.11

tlv4/(iv,

1.44 + 0.07 1.50+0.05 1.56 _+0.08.. ne

R. americana vitellogenin and vitellin melanogaster vitel, 1982), the ovaries horylate the vitel-

ophoresis and isoogenin of R. amerind (S.D Pereira and R. americana vitelphoresis, shows one nd two minor ones ar mol. wts (Bianchi n in the presence of veral protease inhibitors do not modify this pattern • D. Pereira and A. G. Bianchi, unpublished suits). The relative intensities of the vitellin fracms are somewhat variable and, in some preparams, practically only fraction 3 is present (see Fig. I of Pereira and Bianchi, 1983). Isoelectric focusing ' vitellin gives only one defined fraction with an )electric point equal to 6.4 (Pereira and Bianchi, 83). These results indicate that, unlike some other sects which have more than one type of vitellin aaboden and Law, 1983; Adams and Fillipi, 1983; flly and Telfer, 1977; Mintzas and Kambysellis, 82), R. americana has only one vitellin, as was ggested in our original description of this protein ianchi et al., 1982). The vitellogenin and vitellin of R. americana are made up of the polypeptides V~, V2, V 4 and V 5 ianchi et al., 1982; Pereira and Bianchi, 1983). (Bianchi mited proteolysis of these polypeptides shows that Limited each ch type has a specific cleavage pattern indicating at they are not merely derived from adventitious that proteol.~ oteolysis in the samples as suggested by Imboden d Law (1983). Furthermore, extraction of vitellin and in the presence of several protease inhibitors did not modif' adify the subunit pattern of this protein (S.D. reira and A. G. Bianchi, unpublished results). Pereira Lp O+ rophoretic analysis and Both bidimensional electro limited proteolysis of vitelloggenin and vitellin point to Le individual subunits in the structural identity of the :s further imply that the the two proteins. The results technique utilized to extract• vitellin does not introation of the subunits of duce any detectable modificati< this protein. Finally, the present ;sent results require that the relative numbers of the individual polypeptides (VI, V2, V 4 and V~) in native vitellogenin and viteUin must be the same. In view of the above results ults, it is clear that the differences in the mol. wts and isoelectric points itellin of R. americana between vitellogenin and vitellin zations in either the mol. cannot be ascribed to modifications wts and isoelectric points of~f any of the individual these differences cannot proteic subunits. Likewise, these arise from differences in the number of subunits present in the native molecules. cules. The results thus imply that the differences bet~ etween vitellogenin and vitellin are a function of their fir non-proteic constituents, i.e., lipids and/or carbohhydrates. In this regard, the lipids are more likely candidates than carbore covalently linked to hydrates since the latter are polypeptides and differences resulting therefore should also have been observeq ced in the bidimensional electrophoretic analyses of the vitellc linic polypeptides. Differences in the 1 vitellogenin and vitellin are know +..,

. . . .

] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

r

. . . . . . . . . .

Locusta migrat~ osamia cynthia

et al., 1981) and Phyl, 1977).

Acknowledgement from the Fundaq~ Paulo (FAPESP (FINEP), Convr+ No. 401.372/83. about the style of M. H. Juliar 32p.orthophosph~ statistical analysi FAPESP and A. Bioch< Biochemistry De CNPq.

was supported by grants i Pesquisa do Estado de Silo "a de Estudos e Projetos ~1/295/00 and from CNPq [ to Dr F. Quina for advice t; to Drs J. C. C. Maia and ly providing us with r S. D. Carvalho for the is a graduate fellow from i is a staff member of the a research fellow from

r/.~1-

E

aCES Adam 83) Vitellin and vitellogenin Adams T. S. and con ,'sis in the first gonotrophic concentrations cycl tomestica. J. Insect Physiol. 'cle of the ho 29, 723-733. Bianc] A. G. d~ Bianchi L and Terra W. R. (1982) Vitellogenins a~ ~lymph proteins involved in Vite ~ra americana. Inseet Biothe oogenesis chem. chet 12, 177Brenn M. D. a Brennan P. (1982) Phosphorylation o f tthe vitellogq des of Drosophila melanogast "aster. Insect 1 i9-672. Chino H., Yamagata M. and Sato S. (19" (1977) Further characteri: terization of lepidopteran vitellogenin enin from haemolymph and mature eggs. Insect Biochem. 7, 125-131. 1 Chinzei Chinzq Y., Chino H. and Wyatt G. R. (1981) Purification vit from Locusta and properties of vitellogenin and vitellin migratoria. Insect Biochem. 11, 1-7. Cleveland chner M M. W. and Laemmli Clevel~ D•, Fisher S. G., Kirschner U.]K. (1977) Peptide reaping by limited lim proteolysis in sodi sodium dodecyl sulfate and analysi lysis by gel eleetropho: )horesis. J. biol. Chem. 252, 1102-11( 1102-1106. Engelr ;elmann F. (1979)Insect vitellogenin: enin: identification, biosynt 'nthesis and role in vitellogenesis. Adv. A Insect Physiol. 14, 49-107. Hagedorn H. H. and Kunkel J. G. (1979) (197~ Vitellogenin and vitellin in insects. A. Rev. Ent. 24, 47 475-505. of vitellins Imboden H. and Law J. H. (1983)) Heterogeneity Heterc Manduca and vitellogenins of the tobaco hornworm, hot sexta L. Time course of vitellogenin appearance in the Biochem. 13, haemolymph of the adult female. Insect In 151-162. (19 Johnson N. L. and Leone F. C. (1964) Statistics and Experimental Design in Engineering and the Physical Sciences, Vol. 1. John Wiley and Sons Son Inc.. Kelly T. J. and Telfer W. H. (1977) Antigenic Ant and electrophoretic variants of vitellogenin in Oncopeltus Om blood and their control by juvenile hormone. De Devl Biol. 61, 58-61. Kessler S. W. (1975) Rapid isolation of antigens from cells antibody adsorbent: with a staphylococcal protein A an ibody-antigen comparameters of the interaction of antib plexes with protein A. J. Immun. 115, 1617-1624. Koeppe J. and Offengand J. (1976) Juvenile J hormoneinduced biosynthesis of vitellogenin in Leucophaea maderae. Archs Biochem. Biophys. 173, 100 100-113. Lara F. J. S., Tamaki H. and Pavan C. ((1965) Laboratory culture of Rhynchosciara angelae. Am. Nat. 99, 189-191. Marinotti O. and Bianchi, A. G. de (1983i) A larval haemolymph in the eggs of Rhynchosciara americana. Insect Biochem. 13, 647-653. The yolk proteins of Mintzas A. and Kambysellis M. (1982) Tt Drosophila melanogaster: isolation and characterization. Insect Bioehem. 12. 25--qq and O'Farrel P. H. (1977) tal electrophoresis of basic 12, 1133-1142.

A. G. DE BIANCHI and S. D. PEF,EIr 983) Vitellogenin and a: further characterBiochem. 13, 323-332. arini A. G., Pueyo M.

T. and Lara I incubation of., composition o Cult. 28, 654-/

A medium for short-term based on the haemolymph chosciara americana. Cidnc.