Role of the lysine and arginine residues of vitellogenin in high affinity binding to vitellogenin receptors in locust oocyte membranes

aiodlumca ef B¢ofgly~'i~a Acre. !133 11t)92) Ihll- l flb rt:. ]992 ElsevierScience PuhlishersB.V. All rightsre~,erv~dill fiT.4Hst)/tJ2/~nS,011

l (~0

P,BAMCR 131)S3

Role of the lysine and arginine residues of vitellogenin in high affinity binding to vitellogenin receptors in locust oocyte membranes Axel Roehrkasten

and Hans-Joerg Ferenz

bru.v¢ p/tys/o[t~.~. GnJ/tp. Department Biolvg~. LtJfit'(,~if~~o l'Otdetlb~rg. Oldt.nburg ((;erJ,any )

(Received I May 1091)

Key w~lrd.~: Vileikl~enin:Receptor: Endt)cytosis:Ly.~incand arginiae residue;(Oocyrel A specific cell surface receptor mediates the endneytasis of the :yolk protein vitellogenit~ (VTG)t a lipoglycoprotcin, into growing oocytes of the insect Loeusta ndgratoria. The ability of the VTG receptor to recognize VTG was analyzed in Mnding tests a~er modification hy five lysine-specific and two other reagents. Progressive chemical modification of the lysyl and ar~inyl residues resulted[ in reduction or loss of the derivatized VTC to compete for binding to the VI'G receptor with unmodified ~,q'G. Although the precise role of the iysine residue~ in receptor binding remains to he defined we conclude that they are involved in expression of a recognition site interacting with the binding domain of the VTG receptor. Sulfhydryl groups are not involved in the conformation of the recognition site or binding ability of VrG.

Introduction Dtjring vitellogenesis oocytcs of gviparous animals aceumuialc yolk protein precursors, vitelloger~ins (VTG). from lhe hacmolymph or blood b~' receptormediated endocytcrsis-. Receptor~mediated endocytosis is a process in which a membrane-enclosed receptor molecule specifically binds its ligand, the VTG, which is then brought into the cell via coaled vesicles and released to form large yolk spheres. In the insect L o c u s t a m i g r a l o r i a we have recently described the selective blndir~g of VTG to ooeyte membranes [I] and the solubilization and characterization of the locust VTG receptor [2-,J,]. In ligand blotting studies the locust VTG receptor has been identified as a protein with an apparent ntolccular mass of 200 kDa. It is a glycoprotein with N- and O-linked oligosaccharides [5]. The locust VTG is a phospholipoglycoprotcin with a molecular mass of 550 kDa. Its amino acid composition

Abbrcvlations:SP. N st~ccinlmldyt(2,3)propkmate:VT. viteilin: VTG. ~itcllogcnin. Correspondence: I I.-J. Ferenz. Inst2cLPl'~ysiok',gyGro~,p, FB 7. Llrli-

~,t:rsity,P.O. Box 2503, D~2O()00ldt.nburg.Germany.

has been studied [6,7]. The VTG has high contents of aspartie acid, glutamie acid and leucine, and low contents of histidine, methionine and tryotophan. No infiwmation ,,m its amino acid sequence is available, More detailed s~quen~,,e information from other animal species indicate, however, that the VTOs appear to have been conserved during evolution [8]. Further, more, it was recently observed that VTG receptors can recognize and bind VTGs from other species [9,10]. The molecular basis of the specific interaction between receptor and VTG is not well understood. The importance to the binding reaction of positively charged t'eglons oft the locust VTG is suggested by our recent observation that suramln can displace VTG from the receptors, presumably by binding to the cationic sites on the VTG. Trypan blue acts in the same way even precipitating the VTG. On the basis of these results we suggesled that VTG uptake is dependent on the presence of recognition sites [1 1]. To determine whether some amino acid residues of the VTG are of importance for the recognition by the receptor, different amino acids have been selectively modified and the effects of modification have been analyzed, in continuation of a preliminary study [2] we present in this communication evidence for the importance of lysine as well as arginine in the tocust VTG for the binding to the receptor in oocyte membranes.

161

Materials and Methods

Reversible dt'rWvti:atkm of ly.vine re~s';dues by 2,3-~ftmethyhnalcw m*hydrMe The dimcthylmaleic anhydride

Anima~

prl~cedure was the same :~s described for mafeie anhydride [15]. Aliquots of such dimelhylmaleic anhydridemodified VT(; were ~akcn :rod the Ireatmenl was reversed by lowering the pH to 3.5 for 30 rain at 20~C. The reaction was stopped by dialysis against incubation buffer. Reduetive methylati,n. Reductive methylation tff VT(i was perlormed :,t 4°C in 0.1 M borate buffer (pH 9.0) using lhe method of Means and Feeney [161. 2 mg of sodium borohydridc were added to the VTG solutions t 13 mg of VTG protein) followed by additions of 0.5 to 5 ~tl or 37% aqueous formaldehyde mlution (increment size max. 1 /el) over a period of 30 min. Excess reagents were removed by dialysis against incubation buffer.

Locusts (Locusta ndgratori~tl wcrc kept under crowded conditions at 3(l°C with a daily pholoperiod of 14 h, and fed fresh grass, wheat shoots and dais.

Chemicat~ N-Suecinimidyl-[2,3-:~H]propionate (['~H]SP) (I m C i / m l toluene, specific at'deity 106 Ci/mmol} was pu:'chased from Amersham-Buehler (Braunschweig, Germany). Acetic anhydride, 1,2-cyelohexandione, dimethylmaleic anhydride, hydroxylamine hydrochloride, iodoacetamide, maleic anhydride and sodium horohydride were obtained from Sigma (Deisenhofen, Germany). Formaldehyde solution (37%, w/w) was purchased from Merck (Darmstadt, Germany}.

Chemical modifications VTG was obtained from locust haemolymph and purified as described previously [12]. The extent of lysine modification by N-succinimidylpropionate (SP), acetic anhydride, maleie anh).dride or rcductive methylation was determined by the method of Habeeb 1"13] with the reagent 2,4,6-trinitrobenzenesulfonie acid as the difference in values obtained for modified and unmodified VTG. The results in the presence of denaturating reagents such as sodium dodecyl sulfate or KSCN before addition of 2,4,6-trinitrobenzenesulfonic acid showed no significant differences. Propionylation. Propionylation was carried out as described previously [1]. For each milligram of VTG protein to be modified, 0.001 to 1.97 p, mol SP were added. The SP needed for this reaction was prepared according to a previously described method [2]. Acetylation. VTG was acetylated by the method of FraenkeI-Conrat [14]. In a typical preparation, I ml of VT(i (12 m g / m l ) in 0.[5 M NaCI was mixed with 1 ml of a saturated solution of sodium acetate with continuous stirring at 4°C. For each milligram of VTG protein to be modified, 1.7 to i3.7/stool of acetic anhydride were added, but distributed over multiple small aliquots (2 tt[) over a period of 1 h with continuous stirring. The pH of the solutions was maintained by addition of 1 M NaOH+ The reaction products were isolated by dialysis against incubation buffer. Maleylation. VTG was modified by maleic anhydride, using the procedure of Glazer et al. [15]. Ratios of 0.31)-12.15 /stool of maleic anhydride (0.02 M in chloroform) for each mg of VTG protein were transferred to glass tubes and the solvent was evaporated by a gentIe stream of nitrogen. V'FG (,5-10 m g / m l in 0.1 M sodium phosphate, pH 8.5)was added with continuous stirring at 4"C and the pH was maintained at g.5 by the additions of 1 M NaOH, The reaction products were isolated as above.

Rere~ihle t&,rieatirario~l of argblb:e side chabts b), 1,2-o'ch~hexanedione. Modification of arginine sldc chains was done as described by Dietl and Tschesche [17]. The VTG ( I I m g / m t of protein) in 0.t5 M NaC1, 10 m M sodium borate (pH 8.1} were diluted with 0.2 M sodium borate (pH S.D, which was either t1.15 M or 0.015 M, respectively, [n 1,2-cyclohexandione. to lt.~ee ti~r~'~,~the origimd volume and kept at 35°C for 2 h. ThE reaction was stopped by dialysis against incubation buffer. Reversal of protein modification was achieved by extensive dialysis of aliquots of cyclohexandione treated VTG against I}.5 M hydroxylamine in 0. I. M mannitol (pl-I 7.01 at 4°C and by incubation of the dialysates at 35°C for 8 h. Redaction and alkylation o f IOG. This reaction was performed according to W¢isgraber et al. [16]. VTG in 0.2 M phosphate buffer {pH 8.0) was reduced by treatment with ~-mt:rcaptoethanol (1 ~zl per mg of VTG protein) at room temperature for 4 h. Iodoaeetamide (15 to 30 mg per 9 mg of VTG protein) wad added to the reduced VTG and the mixture was allowed to reae! for 30 rain at 4~C in the dark. The reaction products ~ere purified by dialysis as usually.

Assays The binding of [3H]propionyI-VTG to the VTG receptor in isolated locust Oocyte membranes was mcasur-~d exactly as described previously [1]. in all binding assays 60 ~.g of oocyte membranes were incubated with 10 leg of [~H]propionyI-VTG (specific activity 95 d p m / n g ) . The chemically modified V'TG was tested for its ability to compete for binding to the memhrane bmmd VTG receptor in the presence of radiolabelled VTG.

Other analytical methods Protein concen'crations were determined by using the Lowry protein a,~say with bovine serum albumin as standard. The concentration of the differenl prepara-

162 tions of modified and unmodified VTG are given in terms of the protein content of the l/pogiycoprotein complex, Molar ratios were calculated assuming a total molecular weight [or the protein a t VTG of 422400 (76.8% of 550000) and a ratio of 206 real of lysine and 147 mot of arginine/mol of VTG [6]. Control and modified proteins were also characterized by agarosc gel eleelrophorcsis in Tris-barbiturate buffer (OH 8,6) (LKH Application Note 310). Resu!ts

To determine whether amino acid residues of the VTG moIecuIe have a functional significance for its binding to the VTG receptor imbedded in oocyte membranes several different amino acid residues have been selectively modified. The types of modification and the reagents used are listed in Fig. 1, The modified VTGs were tested .for lheir ability to compete with radiolabelled V T ~ for binding to the VTG receptor in ooeyte membrane preparations.

Chemical modification of am#2o groups Propion~lation. SP specifically interacts with the eamino groups of lysine residues resulting in a neutralizatiou of the positive charges. Agarose geI eleetrophoresis indicated that treatment with smatl amounts of SP did not affect the migration of the VTG while con,;eutratlons of 50 renal/rag VTG or more increased its mobility due to the destruction of positive charges at the lysine residues (Fig, 2). Binding studies with SP-treated VTG demonstrated that the blinding ability of the modified V T G was reduced or lost after exten-

Reagent

Residue jnadified

ii tl it

e ii 1! t it t ti 0

0

0

0

0

0

0

0

O

0

0

A

B

C

D

E

F

G

H

I

d

K

Fig. 2. Agaro~e gel el~ctrophoresis tat" ttnlreated VTG (lane A) and VTG chemically m~xlified with increasing concentrations of SP: lane B, I am~l/mg; lane C, 5 ~wc2,/ml;; lane D~ 11 afoul~rag: lane E, 27 nmol/mg; lane F, 49 areal/rag; lane O, 99 nmol/mg; lane H, 53.5 nmol/rag; lane I, 987 nmol/mg; lane J" 1.g7/zmrd/mg; lane K. 3.9~ ,umol/mg, The VTG concentration in each lane was 34-37 #g,

sire propionylatiun (Fig, 3). While up to I0 nmol of SP did not affect the binding of VTG to its receptor larger concentrations reduced the specific binding in a dosedependent manner. At a concentration of about 0.5 /~mol S P / m g VTO the ability to compete for binding to the VTG receptor was nearly completely abolished. In our binding studies we used VTG radiolabeled with [3H]SP. The labelling reaction was carried out at a concentration of I amp/ r'3H/SP per mg VTG, a concentration that is far from affecting the reactivity of the VTG. Acetylution. Acetic anhydride interacts with the e. amino group of lysine resulting in a neutralization of the positive charges. Similar to propionyI-VTG, ace~IVTG had a higher mobility in agarose gel electrophoresis (Fig, 4, lane D). Acetylation with 1.71 #tool acetic anhydride per rag V T G resulted in a modification of about 50% of the lysine residues. Such modified acetyI-V'ro was practically unable to compete with

Strucfurat modification ~

L~'5

O -NH-~-CHz-[H]

Acetic ani..ftlride

Lys

0 -NIt-['-CH~

~laleic anh?~ride

Lys

-NH-r-cM :ca-[,o_

N- r,;~ccinimidylproplonaI

|

9,

~. O6 g 05 ~o

~ o3 o cp, o Drmethylmaleic 2.n~y¢4de

Lys

-NM-E-C=[-C~0

Reducfive methyLafi~n

kys

-~lh~-lCH~

Cyclohe~taneclione

Arg

-NH-C.~H O

~e ~ucti,m/alk vlalizi~

Cys

-S-CH=-C-NH z

~NH

Fig. I. Reagems used for Ih¢ chemical modificution nf amino ~cid residues of VYG and the resulting structural modifications.

[o2 010

025

050

075

rnQ untabetled vlt@ttogen~n/rube FiE, 3. s\bitity or untreated VTO tJ,) and unlahelled V'TG propiony-

tatec! v,iCi iI nrnot {[3), 27 nmal (O1 4q nmmol (z~), g9 nmol to) and 535 nmol ~,vt 5P to eumpete with [-'HlSP-lubeJ]ed V']'G [or binding to the VTG l'eeeptnr in lucusl oocyte membranes. ]ncuba|ion was for 60 rain at 30'C in Ihis and all following experlm~nts.

163

I tie

I

i leO

, ioio

o

t~

t~

0

0

0

0

0

0

0

0

0

0

0

0

0

B

C

O

~:

F

G

H

I

J

K

L

M

N

O

P

~05'

o A

Fig. 4. Agiirose gel eleclmphoresis ~;f untreuh:d VTG and cltcmkldly modlf~ca VTG. l'or ,dl Vl Ci mcttifications appropiat¢ conlrllls v+crc m a d e Urlll'~uted ~¢Tl~i.lant~ A: untreated VT, lan~: 1~1a~elic itil~lydride: lane C txmlrol, laat: D 1.171 F.mol/n~g V'I-G; mah:ic anhydride: lane E control, tam, F 2,43 Fmo]/mg VTG; f~lrmaldehyde: hme G 2.04 ttmol/mg VTC-. l:lne H conllld; dlme'hylmaleic anhydride: rt,wer~a]of r~ndification with 1.33 p.mtrl/mg VTG. lane .] 1.33 /~mol/mg VTG, lane K conlr~h iodoacelamid: lane L 17.tJl)areal/me VTG, lane M conlrol: crch~hex~nedion¢: lane N rever,~al oi treat mcnt with 27.27 ~mol/mg VTG, lane O 27.27/~mol/mg V'I(;. lane P control. The V'TG concenb'alJon in each lane was 26 41')/~g.

native [31-1]SP-VTG for binding to the VTG receptor (Fig. 5). Maleyiaiion. R e a c t i o n of maleic anhydride with lysine residues of the V T G molecule results in the placement of an anionic carboxyl g r o u p in place of a cationic amino group. Thus, to obtain the same increase in electrophoretle mobility as with propionyl- a n d acetylV T G only half of the lysine residues n e e d e d to be modified. With 2 . 3 4 / z m o l maleie anhydride 34% were modified. Such maleyI-VTG m i g r a t e d slightly faster t h a n the o t h e r r.lodified V"l'(]s (Fig. 4, lane F). In competition e x p e r i m e n t s this maleyl-VTG failed to inh i b i t [~H]SP-VTG binding {Fig. 6). Only modification

with maleic anhydride concentrations

near 0.5

p, m o l / m g V T G had no effect on b i n d i n g ability. The larger concentrations of m a l e i c a n h y d r i d e n e e d e d for modffic~.tion are due to the high sensitivity o '.,his chemical to hydrolyze to m a l e i c acid.

to6

F

~03

e C2~

010

025 050 lcj u~{~l~eLLe~ wt~iiogemo/i~be

075

Fi~. h. Ahility o i unlrqutvd V-I'~ Io) a n d unlui~eli~d malc~.q-V'l'(.; r e a c t e d wiTh 11.49 ~ m , fl ¢-'A, 03)7 u i ' n , m , ~ ( ,~ }. I.q4 ~ n ~ ! ( ~ ) a n d

2.43 um,il (~31 m¢dei¢ anhydride to compele wi!h i'H~SP-labcllcd v-I'rG tk~r binding to the VTG rccepmor i'~ It~custochre mcmhranem

Dimefhr'hnaleylatio,. Chemical modification with dimethyl maleic anhydride bad the same resuIt as v, ith male[c anhydride except [hat this modification was reversible. A concentration of 1.33 / x m o l / m g V"fG considerabl~, inhibited the ability of the V"FG to cam= pete for b i n d i n g to the V'i'G receptor (Fig. 7). How~ ever, after incubation at pH 3.5 at 20~C the dimethylmaleyI-VTG fully recovered Its binding activity was indistinguishable from the controls. Agarose gel .:l~ctrophoresis ¢xperiment:~ s u p p o r t e d this result: dim,:t', ylmale:,l-CTG moved taster than u n t r e a t e d V'I'G J.ue to the neutralization of the positive charges at lysine residues, while after reversion of the dimethylmaleylation the V T G m i g r a t e d as the u n t r e a l e d control (Fig. 4, lanes J and I respectively).

............

~o.s o

g o~ T-, O2 01

=-01

]

0

I ~

o

010

I

L _

0.25 050 m!~ unlabetled v.=teL~,ocJ.=n~=~ i tube

075

Fig, 5. Canip¢litJv¢ inhibititm =ffthe binding of [JH]SP-VTG by V1 G modified by acetic an~l~,drid¢. Ontrealed '¢TG (t); VTG modified with 1,71 ,.,~mol/mgof acetic anhydride 1~3).

I

I

01'0

I

025 05(3 0"/5 mcJ uHlabeted vll'~l[ug~nm/tub~ Fig, 7, Abflii~of untrcaicd V T G (o) and V T G modified ~ilh dlmelh ylmalelc anhydrlde (I.~3 .~mol/m~, VTG. A) to compete wilh [~HISP-VTG for bindin:~ to the V'~'G receptor in oocyte ~,emhranes, "~T'.e modification was reversed by towering li~¢ ~.]1 t¢~ 3.5 ( 4 ) : the COI1LTO[ II~TG kk'i~l~llf r o ; l i e d

Ill t h e ~3nie w a y (z~F,

164

I .~05'

~o~ ~o~ b. 01

~01 m

r

010

I

....

I

I

025 050 mg unIabelle~ v~t~ll~gemn/tube

I)i0

075

Fig. 8. Ability of unlrcuted V'rG t t,] and VTG tnt,'.dlfied by reducti'~e mclhylation tc~ comocle with [~HISP-VTO for binding to ]ot?lLc,l oocyte membrane.~, Formaldehyde c~mcentrations used were: 0.51 p~mol/mg VTG (z~); I,II2 v m n l / m g V T G (I.2); 2,04 ~.mol/mg VTG i©),

Reductit'e methylation. Reductive methylation is an additional method with which the role of lysine in the binding region of the VTG can be analyzed. This method does not niter the charge of the lysine (16]. Conditions for methylation were such as to avoid negative effects of the treatment of VTG with borohydride, a reagent which might destroy disulfide linkages. Sodium borohydride alone did not effect the binding activity of the VTG (dala not shown). However, the addition of formaldehyde abolished the binding aetivily of the VTG (Fig. 8)~ A formaldehyde concentration as low as 0.5l /zmol/mg VTG, where only 15% of the lys'ne residues are modified, deafly reduced the ability to compete for binding to the VTG-receptor. Modification of 48% of tbe lysine residues with 2.04 ~ m o l / m g resulted in a complete loss of the binding activity. As expected in a~arose gel electrophoresis the migration of the methylated VTG was not affected (Fig. 4, lane G), Chemical modification o f arginine groups The reaetlon of 1,2-cyclohexandione with the guanido group of arginyl residue.~ has been found to be specific and reversible [17-2fl], We used cyclohexanediane to modify the arginyl residues of the locust VTG and the effects of this modification upon competitive binding of VTG to locust oocyte membranes was assessed. Treatment of VTG with 2.73 ttmol/mg resuited in a considerable inhibition of the ability of this protein to compete with [sH]SP-VTG for binding to the VTG-receptor in oocyte membranes. With 27,27 t~mol/mg the binding activity was nearly fu|ly abolished (Fig. 9). Binding activity was restored to tile modified VTG by removal of the cyclohexanedione. In agarose gel electrophoresis the cyelohexanediune-

I

I

0Z5 050 rng unlat:etted ~tettogen,n/tut~P

I

075

Fig. 9, Changes of the llbility of erll'rea~ed V T G ( t ) and VTG mudified ~vith 1,2-cyelohexanedJone (~,. 2.73 / t m o l / m g V'FG; II. 2 7 , 2 7 / c r e e l / r a g V T G ) and V T Q from which the cyelohexanedione h~ld been removed (O. unmodined control; after lrealment with 2.73 /~mol/mg (ca) and 27.27 p . m o l / m g I ra] respeelive]y) to compete wflh [3HISP-VTG for biliding ttt Iocusl oocyte membranes. Methods were as described in Materials und Methnd~,

treated V'I-O migrated further towards the anode (Fig. 4, lane O) than the untreated or regenerated VTG (Fig. 4, IaEc N).

Chemical modification o[ sulfhydryl- and disul[ide groups Reduction of VTG with ~l-mercaptoethanol followed by alkylati0n with iodoacetamide had no effect on the ability of the modified VTG to compete for binding to the VTG receptor in oocyte membranes (Fig. 10). Furthermore, the migration of this modified VTG in agarose gel electrophoresis was not changed (Fig. 4, lane L), Apparently, sulfhydryl and disulfide groups of the VTG are not involved in the binding to the VTG receptor.

,~ 0 5' ~ 0L. = a~03 ~5 ~ 02 H 01 ~

- .... I 070 0

I__ ~ I 025 . OSO ~ q unIabPIl~d v d e l l o g e n l n / t u b e

075

Fig. 10. Ability of tmlreated V T G (*) and r e d n e e d / a l I o l a t e d V T G [ ~ , 8.95 ~mol iodoa~etamide/mg VTG; n , 17.90 ~ m o l iodoacetarnide/nlg V T G ) ltr c'¢.mp~'tg with ['~H]SP-VTG [or bif~din~, to Ihc V'['G reccplor in locust o o w t e membranes.

165 Discussion Previously we observed that negatively charged compounds like trypan bloc or suramin strongly bind to locust VTG inhibiting its binding to the rec¢ptor and its uptake by oocyles [ i 1] and even dissociate rcceptor~ ligand comp[e×es r.uggesting the appearance of positive charges at the VTG molecule [4L Our present ~tudy indicates the presence of a specific cationic recognition site at the VTG molecule for receptor binding. It appears that the presence of lysinc and arginine is required at this ske for the interaction of locust VTG with lhc locust VTG receptor. The VT and VTG. respectively, of Locuxta migratona have a rather high contents of aspartlc acid, glutamic acid and leucine bu! a low contents of cysteine, histidine, methionine and tryptophan (Table I) [6,7]. From the 3750 amino acid residues calculated per molecule of the locust V T / V T G only 206 are contributed by lysine and 147 by argirfine. Depending on the method for chemical modification the binding ability of VTG is completely abolished after modification of 70 to 103 lysine residues (after maleylation: 34%, after reductive methylation: 48% and after acetylation: 50% of the lysine residues). For first indications of a reduction of VTG binding much less lysine residues need to be modified: by reductive methylation 15% = 31, by maleylation 9% ~'_9 and by propionylation less than 5% - 10. For arginine residues we obtahted similar results. For chicken VTG it has been shown that the methylation reaction TABI.E I Amino acid cvmlnxiitian of locust I/F~ VTG Calculations are based an a molecular mass of 550 kDa of which 76.8% are aralao acids [2]. Locust VT and VTG w,~rc ~utntd to be bioehcmically nearly identical [3], Amino acid

Lysll)e Hislkline Argiaine Aspartie acid Threoninc Sefine Glutgrni¢ acid Ptoline Olyeine Alaaine Valine Melhianifle lsolcucin¢ Leuein¢: Tymsinc Phenylalanine Half-cystine Tryploph~n

Molecular mass" 128,17 137.14 17&2O 133.10 119A2 105.09 147.13 115.13 75.07 89.09 I 17.15 149,21 131.18 131.18 181J9 165J9 120.15 204.23

Mai %

5+6 1.5 4.0 11.0 48 7.5 12.6 5.9 5.0 ~.4 7,7 1.7 5. I 10.I 5.11 2.0 1.11 2.2

" Molecularwgightot anhydrousamino acids.

No. or residues 205.8 55. I 147.11 404.2 176,4 275.6 463.0 216.8. 183.7

3118.7 282.9 62.5 t87.4 37!..I 183.7 106.6 3~.7 80.o

does not affect the polypeptide pattern of the modilicd tile chicken V T O {25]. Wc used very low concentratioz:, of the modi~ing chemicals to prevent any protein

degtaaation. In most c×perlmcal.~ we found a dose-de t~endent reduction of the binding ability of the locust VTG. In dimcthylmalcylated locust VTG the binding activity couId cvcn fully be restored indicating that the VTG molecule was not damaged by this treatment. The precise role of both lyslne and arginine in the VTG/rcccptor inlcracti~n remains 1o be determined. In rescmblancc to ~,hc low density lipoprotcin the following possibiIities for the effect of the modifying agents can be suggested [2[]: (a) the positive charges are directly affected or not available because of the presence of the modi~ing agent. However, in this ease reductive methylation should have had no effects since it does not alter the charge and causes no steric hindrance [1@ (b)"lhc modification may affect the chemical rcactivi~ of guanido or e-amino groups independent of charge+ It is known that in certain cnzyme~ lysine residues can form a Sehiff base with substrates 122.23~. (c) The modification affects the conformation of the recognition site either changing the spatial distribution of key lysine or argininc rcsldues or preventing other residues from reacting with the receptor. Similar explanations have been considered for the function of active centres of enzymes containing lysine and argininc residue.~ and reacting with anionic substraws [17,19,20,241. Recently, it has been shown !hat also chicken VTG modified by reductive mcthylation tff lysine residues failed to bind to the chicken oocyte membrane VTG receptor i~ competition experiments with unmodified VTG [25]. Chicken and other V']'G receptors seem to be biochemieally and functionally very similar to lhe locust VTG receptor [26]. These observations and our study are in close agreement with similar comprehensive data reported for the binding of the aPolipoprotein B in human low density lipoprotein to its receptor in mcmbranes of fibroblasts (ReL 21; for reviews see Rots. 27 and 28). For the low densily [ipoprotein r.'zcptor it has been found that its binding domain contains ~steine-rieh regions forming a cluster of negative charges presumably complementary to the recognition site at the apolipoprotein [29]. Also, the human and bovine growth hormones contain lysine residues which were found to be essential for binding to tl~e hormonal receptors [30]. In agreement with the cited reports we conclude that the interaction of VTG with its receptor is probably not simply the result of the presence of certain positive charges, We rather assume that there is a certain spatial clustering of amino acyl residues comprising the recognition site. A cunformational change in the tertiary structure of the VTG appears to occur in response to charge modification of specific, critical lysyl and arginyl residues. However, sulfhyda3'l groups are not involved in the establishment

166 o f t h e c o n f o r m a t i o n since c y s t e i n e - c y s t i n c m o d i f i c a t i o n did n o t zbolish t h e V T G b i n d i n g activity. It s h o u l d also b e c o n s i d e r e d tha'c lipids a n d o f i g o s a c c h a r i d e s m a y p r o m o t e t h e f o r m a t i o n o f t h e r e c o g n i t i o n site: oligos a c c h a r i d e m o d i f i c a t i o n o f t h e Blattella V T a f f e c t e d its u p t a k e by o o c y t e s [31]; locust V T h a s a d i f f e r e n t lipid c o m p o s i t i o n t h a n V T G [7] a n d is less r e a d i l y i n c o r p o r a t e d t h a n t h e n a t i v e V T G [12]. Acknowledgement T h i s r e s e a r c h has b e e n f a c i l i t a t e d by a f i n a n c i a l grant from the Deutsche Forschungsgemeinschaft (Fe 134/51.

References 1 Rochrkaslcn. A. and Fereni~, H. J. (1986) InL .I. Inuert. Reprod. 10. 133 142. 2 Roehrkastcn. A. and Ferenz, H,-J. (1°,861Bioehim. Biophys. Aela 8617, 577 582. 3 Ferernz. I-I.-l. (1990~1 in Ad'-'ances of Inver'lebrate Reproductio~ Illoshi, M. and Yarnashita, O., ads.), Vol. 5, pp. 103-108. Elsevier, Amsterdam. 4 Rochrkasten, A.., Ferunz, H.-J., Hafer, J., Buschmann-G'cbhardt, B. (1980) Arch. ]o~¢l Biochem. Physiol. 10, 141-I49. ,~ I-lafer, ,L, and Fcrt:nz, H.-J. {1991) Verb. Dtsch. Zoo]. Gas. 84, 228. 6 Chen. T.T., Strahlendorf, P.W. and Wyatt, G.R. (1978.1 J. Bir~L Chem. 253. 5325-5331. 7 Chinzci, Y., Chino, H. and Wyatt, G.R. (19811 Insect Biochem. 11, I-7. 8 NardcllL D.. Gcrber-Huber, S, Van her Schip, F,D., Grubcr, M., Ab, G. and Wahl(, W. (19871 Biochemisll3.' 2fi, 6397-64D2. 9 Slii'ani, $., N[ntpf. J. u~d Schneitl~r, "dr. (19901 Jr Biol. Chem. 265, 882-888. l0 I-'laJcr, J., Fischer, A. and Feronz, H.-J. (1991~ J. Cr~mp. Physiol. B (in pres~l.

I] Rnehrkaslen, A. and Furenz, H.-J. (19871 Roux's Arch. Day. Biol. 196. 165-172. 12 Roehfk~sten, A. and Fcreoz, H.-J. {1985) Roux's Arch. Dcv. Biol. 194,411-416. 1,3 Halbceb. A,F.S.A. If19661Aaul, Biochcm. 14, 328-336. 14 FraenkeI-Conrat, H. [1975.1 Methods Enzymol. 4, 247-269. 15 Gla~e~, A.N., Elelange, R.J. and Sigman, D.S. (19751 Chemical Moflifienr~on ok" Proteins, pp. 1 205, Elf;crier Biomedical Press, Amslerdam. 16 Means, G.E. and Feeney, R.E. (19681 lilioehemislry 7, 21~2-22[11, 17 Dicll, T. and "rschcschc H. (19769 Huppe-scylcr~s Z. Phy~iiol. Chain. 357, 657-665~ |8 Mahlcy, R.W., ]nnerarity, "I'.L., P[las, R.B., Weisgraber, K.H.. Brown, J.rl. arid G~ss, E. (19771 .I. Biol. Chem. 252, 7279-7287. 19 Paflhy, L. and Smith, E.L. (19751J Biol. Chem. 250, 557 564. 217 Panhy, L. and Smith, E.L. (19751 L Biol. Chem. 250. 565-569. 21 Weiagraher, K.H., Innerartly, T.L. and Mahlcy, R.W. (197~;) J. Biol. Chem. 253, 9053 9062. 22 Grazi, E, l~,]eioche+H., Martinaz, 13,, Wood, W.A, and Horeckcr, B.L. (1963) B]oehem. Biophya. Res. Common. If1, 4- I0. 9_.3 Fisher. E.H. {196¢) in Structure and Activity of Enzymes (Good, in. 'LW., Harris, J.l. and [[arlley, B.S., ads.), pp. I ( ] -t2tl, Academic Press, London. 24 Riordao. J.F., McEIvany. K.D. and Borders. CL. (10771 Science 195,884-886, 25 Stifimi, 5., George, R. and Schneider, W.J. (19881 Biochem. J. 250, 467-475. 26 Feren•, It.-L (199(t) in Molecular Insect Science (11agedorrt, H.H., Hildebrand, J.G.. ~dwel], M.G. and Law, J.H., eds.l, pp. 131-138~ Plenum Press, New York. 27 Brown. M.S. and Goldstein, .l.L (19861 Science 232, 34 ~.7. 28 Schneider, W3. (t9891 Biochim. Biophy~. Aeta 9lt8, 3fl3-317. 29 5uedhoff, T.C., Goldstcin, J.L., Brown, M.S. and Ras~c], D.W. (19851 Science 228, 815-8.22. 30 De In LEo~a, P., Chene, N. and Martal, J_ (19851FEBS Lett. 180, 295-29~. 31 Goeho¢o, C.H., Kunkel, J.G. and Nordin. J.H. (1988) Arch. In,eel Bioehem. Phy~;iol. 9, 179-199.