Radioimmunochemical studies on 7.8S and 5.7S duck immunoglobulins in comparison with Fab and Fc fragments of chicken IgY

DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY, Vol. 8, pp. 131-139, 1984. 0145-305X/84 $3.00 + . 00 Printed in the USA. Copyright (c) 1984 Pergamon Press Ltd. All rights reserved.

RADiOI~IUNOCHE~ICAL

STUDIES ON 7.8S AND 5.7S DUCK INNUNOGLOBU-

LINS IN COIdPARISON WITH Fab AND Fc FRAGI~NTS OF CHICKEN IgY

Dietlind H~dge and Herwart Ambrosius Laboratory of Immunobiology, Section of Biosciences, University, DDR-7010 Leipzig, G.D.R.

ABSTRACT

Karl Marx

In ducks two forms of low molecular weight Ig's +~, ~ 7.8S and 5.7S, have been described. To compare their antigenicity with Fab and Fc fragments of chicken IgY a double-antibody RIA technique was used in which the binding of 125I-chicken IgY or 125I-chicken IgY(Fc) to rabbit and carp anti-chicken IgY(Fab) and anti-chicken IgY(Fc) antibodies, respectively, was inhibited by duck 7.8S and 5.7S Ig's. Chicken IgY and duck 7.8S Ig (IgY-like protein) are highly crossreactive v~th respect to their Fab as well as Fc part determinants. On the other hand, the duck 5.7S Ig shows nearly identical determinants to the Fab fragments of 7.8S Ig, but a Fc part is lacking. Therefore, we conclude that the 5.7S Ig molecule in ducks doesn't represent a separate Ig class. It consists of the same principal type of heavy chains as the 7.8S Ig, but the H chain of the 5.7S Ig lacks very likely the last two homologous constant regions. INTRODUCTION

The study on non-manmlalian species Ig's provides interesting data on the analysis of Ig evolution. While Ig~, the high molecular weight Ig, was found in all species, thus far

+) Abbreviations used: Ab, antibody; BSA, bovine serum albumin; ChIgY, chicken IgY; IFA, incomplete Freund's adjuvant; Ig, immunoglobulin; 2-NE, 2-mercaptoethanol; M r ~ relative molar mass; RIA, radioimmunoassay; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 131

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investigated, with similar antigenic and structural properties like mammalian I ~ , the low molecular weight !g's show a v~der heterogeneity with respect to the occurrence of a higher number of isotypes. The most interesting aspect in this direction is the fact that the low molecular weight 7S Ig's of birds, reptiles, and anlphibians often called as igG-like Ig's in the literature are not antigenically related to IgG of man~als (I-4). Therefore, it is better to designate the 7S ig's of birds and lower vertebrates as !gY-like proteins in accordance to the term "IgY" proposed for the chicken 7S Ig by Y eslie and Clem (5). In some birds, like ducks and geese (2,3,6,7,8,9) and reptiles (10-12), like turtles and tortoises, a second low molecular weight Ig of 5.7S and with H chains of 35,000 Da could be shov~ v~ich is antigenically deficient to the ig of igY class. That ~vas analyzed in the case of ducks by Grey (7) and Zimmerman et al. (8) using H chain-specific rabbit anti-duck IgY and anti-Ch!gY sera for qualitative Ouchterlony techniques. But there no quantitative data about the relation of 7S and 5.7S Ig's until now. In this paper we describe the degree of antigenic relationships between the both low molecular weight Ig's of ducks to ChlgY and its Fab and Fc fragments using anti-Fab and anti-Fc antibodies. ~TERIAI, S AIYD I!ETHODS Animals. Domestic chickens (Gallus ~allus domesticus L.) were used as normal serum supplier for tile preparation of IgY fraction and its papain fra~uents and domestic ducks (Anser plat,yrrhynchos domestica L.) for the isolation of purified antibodies against BSA of 7.8S and 5.7S types. For this reason, ducks vlere intrs~uuscularly imz~unized to BSA emulsified in !FA according to the method of Grey (7). Blood was collected eight days after the first 25-rag-dose injections or ten days after the last immunization step vfnen ducks were hyperii~nunized. Preparation of l~'s. ChlgY ~vas prepared as described previously (2). Iklck 7.8S and 5.7S Ab's to BSA ~vere isolated from ir~une sera by an irmuunoadsorbent technique using BSA cross-linked by g!utaraldehyde (13) and g!ycine-IICl-0.15 N ITaC! buffer, pH 2.8, for the elution of specific antibodies from the adsorbent. Finally, several gel filtration steps on Sephadex G-200 (Pharmacia AB, Uppsa!a, S~veden) were carried out for t h e separation of the 7.8S from the 5.7S fractions. Papain fra~lents of Chl~oy. i~ab and l~c fra~nents vfere obtained by digestion of I~ ~ ~mol~ papamn accordmng bo Porber (14) but v:ith the exception that we worked at a enzyme-protein ratio of I : 50 (w/w) and a shorter incubation time of 6 h at 37°C. The separation of the tvzo fra~uents v~as done by starch block electrophoresis (0.05 ~ borate buffer, pll 8.6; 26 h; 4°C; 600 V/m) and by gel filtration on Sephadex G-200 (0.01 I~ Tris-HCl buffer, pH S.O, containing 9 g HaCI and 0.2 g HaIT3/I. By SDS-PAGE analyses the fragments showed Ilr'S of 40,000 Da (Pab) and 52,000 Da (Pc), whereas the last consisted of units of 26,000 Da, if the samples were reduced and alkylated v:ith 0.2 I~ 2-~E and 0.25 ~ .

"A-

•

".

J-

A-

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iodoacetamide, respectively, before the electrophoresis u~as started (15). SDS-PAGE. The estimation of the Ifr of the different Ig fractions was carried out by SDS-PAGE after Ueber and 0sborn (16) on 5 % gels using intact Ig fractions as well as reduced and alkylated saz~ples (15). Deterr~nation of protein content. It was performed by measuring the extinction at 280 mu by nleans of a U~'-spectrophotometer (VSU iI, VEE Carl Zeiss, G.D.R.) using E of 12.0 for the ChIgY and duck Ig's and of 14.0 for the Pab fra~nent of ChIgY and 10.0 for the respective Pc fraction (at 280 ~ , 1 % solution). Radioinmlunochemical procedures. The double-antibody RIA technique employed ~as described earlier (2,3). Pot this, we used anti-Cl~gY(~ab) and anti-ChIgT(Pc) antisera produced in rabbits and in carp (3). The absorption of anti-fragment sera was made by means of the corresponding papain fragment preparations coupled to ClTBr-activated Sepharose 4B (17). RESULTS Characterization

of used Ab materials of ducks

From ducks in~munized primarily v~th BSA in IPA we could prepare an anti-BSA fraction by means of affinity chromatography consisting of about equal amounts of 7.8S and 5.7S Ab's with traces of I ~ only (Fig. I). By repeated gel filtration steps on Sephadex G-200 the both Ab molecules were purified until immunoelectrophoretically pure fractions were obtained. By SDS-PAGE the Mr'S were estimated v~th values of 169,000 Da for the 7.8S Ab and 120,000 Da for the 5.7S Ab fractions. After reduction and alkylation of the Ab samples Mr[S were determined for the polypeptide chains with 63,000 Da (H chain of 7.8S Ab), 35,000 Da (H chain of 5.7S Ab), and 24,500 Da for the L chains of both Ab types (Pig. I). As standard Ig we used ChIgY ~ h 170,000 Da (intact molecule), 64,000 Da (H chain) and 22,000 Da (L chain) as well as human IgG (150,000 Da) with polypeptide chains of 52,000 and 23,000 Da. In order to prepare pure 5.7S Ab's from ducks in a simpler modus ducks were hyperimmunized with BSA. In this case, in the immune sera this Ab type can be found almost as the only one. Radioimmuno chemical investigations In the first series of experiments we employed rabbit antiChlgY(Pab) and anti-ChigY(Pc) Ab's in order to determine the degree of differences in regard to the antigenic properties of the 7.8S and 5.7S Ab's of ducks in comparison to the ChIgY (Figs. 2 and 3). In both cases we found the expected high antigenic relationship ~et$een the ChIgY and the 7.8S Ig of ducks already described by Zimmerman et al. (8) and our reports (2-4).

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SDS-PAGE on 5 % Gels: (a) ChlgY and its H and L chains; (b) Duck anti-BSA Ab's from primarily immunized animals w i t h 25 mg BSA in IFA collected 8 days after injection (7.8S and 5.7S Ab's);

170,000 -

I

64,000 -

I

O

(c) H and L chains from (b). I

6 mA/gel, 4.5 h, staining of protein bands with ServaBlau G. Anode is on the bottom.

22,000 -

PIG.

O

1

On the other hand, there were significant differences in the capacity of 5.7S Ab's of ducks to inhibit the standard systems. Accordingly, this 5.7S fraction turned out to be a good i~libitor of the 1251-CI~gY~ - anti-ChlgY(Pab) system, but it didn't show an inhibition in the 1251_ChigY(Fc) - an~_-~n o ~ =~l~'~(Pc) ~ system. Besides, in the Pigs. 2 and 3 the specificity of our rabbit Ab's used for the RIA was demonstrated employing highly purified Fab and Fc fraG~lents of ChlsY as inhibitors. In the Figs. 4 and 5 we give Bhe results using the corresponding anti-fragment antisera produced in carp. Here, the experiments resulted in the equal statement represented with the rabbit Ab's. Because the tested inhibition activity of L chains of ChicY is low in contrast to the Fab fra~uent most antibodies in the antisera must have binding specificity for the Fd Darts of investigated Ig's. DISCUSSION Up to now, Grey (6,7) and Zinmei~lan et al. (S) published the only relevant data on the structural, i1~mlunochen~ical, and biological properties of ig's of ducks. They found three Ig types in this species, Ig~ and two lov; molecular vJeight Ig's with 7.8S and 5.7S and H chains of 62,000 - 66,000 Da (7.8S Ig) and 35,000 (5.7S Ig), respectively. Grey (7) could conclude

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% Inhibition

~~u 50

ChlgY

ChlgY(Fc)

] 5.?S Ig ck I gY

Inhibitor (rig 3

FIG. 2 I C u r v e s Zor !nhibi %ion of ,omndln~ of 2 5 1 - C h l g Y to R a b b i t an-~iCl~IgY(~?ab) Ab's: ~he antisertuu vlas u s e d a% a I : 10,000 dilution, 55 of r a d i o l a b e l ! e d a n ~ m ~ e n v~as bound, if no i n h i b i t o r vias added.

',~

-~'~"

=/=

Inhibition

2

16

lz5

1,ooo

~oo

~ooo

Inhibitor (ng)

~IG.

3

C u r v e s for i n h i b i t i o n of B i n d i n g o f , 1 2 5 i - C h l g Y ( ~ c ) to ~ a o b m o a n t i - O h l g Y ( P c ) Ab's: The a n % i s e r u ~ v:as u s e d at a I : g,O00 dilution, 45 :,5 of r a d i o l a b e ! l e d a n t i g e n "~vas bound, if no in/!ibifor was added.

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7.8S AND 5.7S DUCK IMMUNOGLOBULIN

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% Inhibition 2E ChlgY

5~

\ :higY(Fab)~

h

IgY(Fc)

•

igY

10C +,

i

Woo

+~,

~oo

~oo

Inhibitor(ng) FIG.

4

Curves for I n h i b i t i o n of B i n d i n g of 1 2 5 1 - C h I g Y to Carp antiCl~Y(~ab) Ab's: The a n t i s e r u m was used at a I : 1,000 dilution, 60 ~ of r a d i o l a b e l l e d a n t i g e n was bound, if no i n h i b i t o r was added.

:[

% Inhibition k 57SIg ChlaY

~

ChlgYlFob)

5C

~ lb

1~

1,o~o

~

klgY

r,,,hoo

Inhibitor(ng) FIG.

5

Curves for i n h i b i t i o n of B i n d i n g of 1251-ChlgY(Fc) to Carp a n ~ l - C h ! g Y ( ~ c ) Ab's: The a n t i s e r u m was used at a I : 800 dilution, 50 ~,~ of r a d i o l a b e l l e d a n t i g e n v~as bound, if no irahib i t o r v~as added. ~°

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from his studies on the m e t a b o l i s m of the two low m o l e c u l a r weight forms that this 5.7S protein was a de novo synthesized Ig molecule. He couldn't find a breakdov~ in vivo from the 7.8S to 5.7S form by experiments in which 131I-labelled 7.8S protein was injected into normal ducks. However, the possibility that the 5.7S type is a precursor of the 7.8S Ig is not excluded by those studies. But the kinetics of Ab production in ducks v~ith the appearance of Ig~ Ab's at the first few days after the start of inmmnization, the existence of almost equal amounts of 7.8S and 5.7S Ab's after eight days and later with the occurrence of 5.7S Ab's only evidenced a temporally balanced m e c h a n i s m of i ~ u n o r e ~ l a t i o n , at vJhich all three Ig forms play their definite role. Uith our experiments we v:anted to evaluate quantitatively the degree of ~.nomgenmc relationships betv;een %he both low molecular v~ei~ht !g's of duck on one hand and the 7S Ig of chicken, the so-called !gY, dominantly occurring in normal as v;e!l as in i~uune sera, on the other hand. Por this, vJe used anti-ChlgY (papain fragmlent) Ab's produced in rabbits and in carp, tyro phylogenetical!y distant species v£~ich surely recognize different determinants as foreign. Our data shov; that the 5.7S Ig molecule of ducks really lacks a Pc-like part v~q~ich exists in the 7.8S form of duck as u-ell as in C]C_~gY. it can be assumed that the typic C-terminal domains are absent in the 5.7S Ig type v;hen compared ~rmtn ~r and one polypep 0±de ~ ~ructure of our CI~I~I(9c) preparation consisting of tyro 26,000-Da units covalently combined by S-S bridges. The statement that the 5.7S Ig doesn't have Pc part determinsm_ts agrees v:ith the facts that (a) the carbohydrate content vlith a value of 0.6 % is lov~ (S) in opposite to the higher amotu~t in the 7.8S molecule, as 5 % (S) or 6.4 !~ (2); (b) their H chains have a smaller iT • and (c) the 5.7S Ab's v:ere inable to fix duck complement (7~] The high cross-reactivity betv~een the ChlgY and the duck 7.8S Ab shov,m here could be demonstrated already earlier by Zi!~erman et al. (S) using gel inuuunodifiuoion techi~ique~, in previous publications (2-~) v;e have con±turned onao employmng sensitive RIA systems in order to prove above all, in ud~ch ve± oebraoe species an icY-like protein occurs. These e x p e r i m e n ~ have led to the conclusion that all species of birds, reptiles, and anuran m~7_phibians, so far tested, possess a lov; molecular v;eight Ig antigenically related to chicken IcY. That is also in agu~eement u~ith the results of Leslie and Benedict (10) vf~o could find IgY-!ike proteins in the serums_ of Japanese quails and pheasants and to our findings that further avian species, as buzzard, goose, C~.inea-fo~:~l, kestrel, kite, pigeon, and turkey, contain IcY-like !g's in the serunu using the same RiA system v;ith a n t i - C h i g Y ( n a p a i n _~ -~ra~.~eno) = Ab's mentioned in ~0nm'~o paper (unpublished data]. _~

n.

.

.u

ideally, the relationship of two protein molecules should be based upon a~<~N_no acid sequence analyses, hov;ever, the relev<~no data from non-ma~m~alian species are a~ present quite limited. Because the cross-reactions betu~een related proteins to be due to structural sir?~larities, therefore, a convincing

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I

criterion for their classification is tke serological analysis proven very usefully in d e case of rlamm~alian ig classes (19) According to Vaerman (20), there is sufficient justification to regard the evidence of a clear i~muunological cross-reactiviby as first-order criterion as v:ell as a similariby in the primary ~ - -u~_ l .~C ~u~.I '~ ~ e . J- q

•

A C KHOUL E D G E i . ~ E I ] T S We wish to thank [its• C• Pritzsche in the performance of experiments.

for her valuable assistance

± ~z..,J.,r,i~..i,].~ C ~ o

I. }~HTA, P.D., REiCKLIil, H., and T ~ S I , studies of vertebrate il~u~unoglobulins, 1272, 1972.

T.B. Comparative o• mrmuunol. 109,

2. HADGE, D., PiEB!G, H., and AI~ROSIUS, H. Evolution of low molecular weight inmunoglobulins• I. Relationship of 7S in~munogiobulins of various vertebrates to chicken igY. Developm. Comp. !rmuun• --4, 501, 1980 a. 3. HWdDGE, D., PIEB!G, H. PUSKAS, E., and ~iBROSIUS, IT. Evolution of low molecular weight inmunoglobulins. II. 17o antigenic cross-reactivity of human IgD, human igG and !gG3 to chichen IgY. Developm. Comp. ir~un. 4, 725, 1980 b. 4. ~IBROSiUS, K. and HADG~, D. A phylogenetic view of avian ir~unology. Polia biol. (Praha) 28, I, 1982. 5. LESLIE, G.A. and CLEN, L.W. Phylogeny of irmuunoglobulin structure and function. III. In~uunoglobulins of the chicken. J. Exp. ~ed• 130, 1337, 1969. 6. GREY, H.ii. Duck ir~mnoglobulins. !. Structural studies on a 5.78 and 7 . 8 S ~ - g ! o b u l i n . J. !mmunol. 98, 811, 1967 a. 7. GREY, H.~L. Duck immunoglobulins. II. Biological and immunochemical studies. J. Izmunol. 98, 820, 1967 b. 8. Z i I 3 ~ N , B., SHALATiN, N., and GREY, I{.N. Structural studies on the duck 5•78 and 7•88 irmmunoglobulins. Biochen~stry IO, 482, 1971. 9. HIDGE, D., FiEBIG, H., and ~ R O S ! U S , H. Strukture!le Untersuchungen an Immunglobulinen niederer Wirbeltiere. I. Uber die Evolution der niedermolekularen Immunglobuline. Wiss. Z. Karl-~larx-Univ. Leipzig 26, 67, 1977. 10. LESLIE, G.A. and CL~ff, L.W. Phylogeny of immunoglobulin structure and function. VI. 178, 7.58 and 5.78 anti-DNP of the turtle, Pseudam,ys scripta. J. Immunol. 108, 1656, 1972.

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11. CH~tqTRAITD, S.L., LIT}~IAI[, G.W., LAPOII[TE, H., GOOD, R.A., and FROIt[EL, D. The evolution of irm~une response. X!i. The irmnunoglobulins of the turtle, llolecular requirements for biologic activity of 5.78 immunoglobulin. J. Inm~unol. 107 , I, 1971. 12. A~fBROS!US, H. Innmunog!obu!ins and antibody production in reptiles in: Comparative In%munolo~. J.J. I~
gel

17. CUATRECASAS, P. Protein purification by affinity chromatography. Derivatization of agarose and polyacryl~_ide beads. J. biol. Chem. 245, 3059, 1970. 18. LESLIE, G.A. and BEI~DICT, A.A. Structural and antigenic relationships between avian immunoglobulins. I!i. Antigenic relationships of the immunoglobulin of the chicken, pheasant and Japanese quail. J. immunol. 105, 1215, 1970. 19. I~OH, S.H., JAHODA, D.!!., and ROUE, D.S. iz~unog!obulin classes in mammalian species identified by cross-reactivity with antisera to human immunoglobu!in. Ir~m~unochemistry 10, 805, 1973. 20. VAERf~LI~T, J.P. Studies on igA in~nunoglobulins in man and animals. Sintal-Louvain, 1970. Received Accepted

: October, : January,

1982 1983