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An immunoglobulin-binding component from rabbit antisera, bearing Fab antigenic determinants
Immunology Letters, 1 (1979) 147-152
© Elsevier/North-Holland Biomedical Press
AN IMMUNOGLOBULIN-BINDING COMPONENT FROM R A B B I T A N T I S E R A , B E A R I N G Fab ANTIGENIC DETERMINANTS M. J. ESCRIBANO Laboratoire d'Immunochirnie, I.R.S.C.; B.P. no. 8, 94800 Ville/uil; France
(Accepted August 31 1979)
1. Introduction A fraction of apparent low molecular weight called Fill, has regularly been found to be absorbed with the antibodies when rabbit immune sera were allowed to react with Sepharose-bound antigen. This fraction was present in antihapten as well as in antiprotein rabbit sera. It was separated from the antibodies on Sephadex G200, where it was eluted in third position after the IgM and IgG molecules. According to the elution volume on this resin, its molecular weight should be less than 13,700 [ 1,2]. FIII does not combine with the antigen but combines with the antibody [2]. This binding appears not to be related to the antibody activity since Fil I also combines with normal rabbit IgG; it seems, however, to be speciesspecific since among several species tested, namely rabbit, man, beef and chicken, only rabbit IgG showed a reaction with the rabbit Fii I fraction (M. J. Escribano, unpublished observation). This fraction referred here as an immunoglobulin-binding component (IgBC) was shown to be immunogenic in chickens. One antiserum, obtained after a single IgBC injection, precipitated this component in gel but did not, in particular, precipitate the immunoglobulins [ 1]. Thereafter, others chickens were immunized and the results were different since most of them did not give precipitating antibodies after the first injection. Immunization was then pursued and after two or more injections some sera had the particularity not only of precipitate IgBC but also the immunoglobulins IgG and IgM. In immunoelectrophoresis, the reaction with both Ig was indistinguishable from that observed with antisera obtained by immunization with normal rabbit serum (conven-
tional antiserum). These very peculiar antisera were then compared to conventional anti-lg sera and it was shown that both were functionally quite different. This was demonstrated by the following results: (1) anti-IgBC reacted with Fab but not with Fc fragment, whereas conventional antisera recognized, as expected, mainly Fc determinants;(2) absorption with IgBC removed in anti-IgBC sera all the anti-IgG antibodies. On the contrary, in conventional sera IgBC removed anti-Fab but not anti-Fc antibodies. These results indicate that the antigenic determinants which are recognized by anti-IgBC are located in the Fab fragment only and that moreover, those determinants are the same as the Fab determinants which are recognized by conventional sera. On the other hand, anti-IgBC precipitated H and L chains. IgBC behaves thus as one (or perhaps several) IgG fragments located in the Fab piece, in both L and Fd chains.
2. Material and methods IgBCpreparation. IgBC was obtained from either anti-DNP or anti-HGG (human gamma-globulin) rabbit sera as previously described [1,2]. Chicken anti-IgBC sera. Six chickens were immunized with IgBC from anti-HGG serum and 4 with IgBC from anti-DNP serum. In all instances 50-100/ag of IgBC were given subcutaneously in complete Freund's adjuvant two or more times. Each injection was spaced for 3 - 4 weeks. The animals were bled regularly 3 weeks after each injection. Rabbit IgG and IgM. Pure IgG and IgM were obtained by Sephadex G200 filtration of antibodies
147
obtained by irrmmno-adsorption from anti-HGG or anti-DNP sera of the early response (1 week after the first antigen injection) in order to obtain in particular IgM. Chicken anti-normal rabbit serum. Two chickens were immunized with normal rabbit serum, enriched of IgG and lgM prepared as above. Two injections of 0.5 ml enriched serum were done subcutaneously in complete Freund's adjuvant at a one month interval. The serum was collected 3 weeks after each injection. Horse anti-normal rabbit serum was obtained from the Institut Pasteur, Paris. lgBC imrmoloadsorbent. IgBC (a pool of several preparations) was coupled to BrCN activated Sepharose, (Pharmacia, Sweden). About 1 g resin containing 100/ag of covalently bound lgBC was prepared. To absorb anti-IgBC antibodies, the antisera were passed through the resin (which was packed in a small glass column), several times until all antibodies were removed. This was controlled by the measure of fixed antibodies which were eluted between each contact, with acid buffer. Finally the sera depleted o f anti-IgBC antibodies were concentrated in a Minicon Concentrator (Amicon, U.S.A.), until the initial volume was reconstituted. Preparation o f Fab and Fc fragments. Twenty mg of pure IgG prepared as above were digested by papain [3]. After dialysis against phosphate-buffered saline (P.B.S.) the digest was passed through a protein A-Sepharose (Pharmacia, Sweden) column. Protein A retains Fc efficiently and allows collection of Fab in pure grade. Acid elution of the bound material showed that it consisted not only of Fc but also of incompletely digested IgG and of a small quantity of Fab.
Pure Fc was obtained by the Porter's technic [3], (Pure Fc was a gift of Dr. G. Bordenave, Institut Pasteur Paris.) Preparation o f H and L chains. Pure IgG was reduced with dithiothreitol: (6 mg IgG dissolved in 0.4 ml Tris buffer pH 8.2 + 0.6 mg dithiothreitol, 1 h at room temperature), and alkylated with iodoacetic acid: (1.8 mg at -2°C 1 h) [4]. H and L chains were then separated on a Sephadex G75 column equilibrated with 1 M propionic acid [5]. Alternatively, L chain was obtained in a pure grade by adsorbing reduced IgG on protein A-Sepharose which retains H and unreduced IgG but not L chain.
3. Results 3.1. Antibodies in IgBC antisera About half of the immunized chickens formed precipitating anti-IgG antibodies from the second IgBC injection. Other sera did not give precipitin reactions but were able to agglutinate sheep red blood cells coated with IgG. Only precipitation reactions will be considered in this study. In general, no, or only very weak, precipitation was observed with the injected IgBC, the precipitation always being stronger with IgG than with IgBC. Finally the sera obtained by immunization with IgBC obtained from either DNP or HGG rabbit antisera had identical characteristics.
3.2. Reaction o f lgBC antisera with rabbit lgG and IgM The immunoelectrophoresis depicted in fig. 1, shows that anti-IgBC reacts with both Ig, and moreover
Fig. 1. lmmunoelectrophoresis of rabbit lgM and lgG developed with chicken anti-rabbit serum (conventional serum) and antiIgBC chicken serum. Electrophoresis was performed in Agargel (Oxoid, England) plates 10 cm length in barbital buffer pH 8.2 for 1 h under 100 V. IgM and IgG were employed at the concentration of 1 mg/ml. 148
-
~
~
÷
+
A
t Fab
B
D Q Fc A~_-
a n t i - l g (horse) ,, (chicken) - IgBC ,,
Fig. 2. Imunoelectrophoresis of isolated Fab and Fc fragments. The experimental conditions were the same as in Fig. 1. Fab and Fc were employed at the concentration of 1 mg/ml. A and B were developed with conventional antisera. A: horse anti-rabbit serum; B: chicken anti-rabbit serum. C and D were developed with two different anti-IgBC chicken sera.
the image is indistinguishable from that obtained with conventional antiserum (in this experiment conventional serum was chicken anti-rabbit serum).
3.3. Reaction with Fc and Fab Fig. 2 shows that whereas conventional antiserum (horse or chicken anti-rabbit serum), reacts with both Fab and Fc pieces, anti-IgBC precipitates only Fab. 3.4. Comparison in double diffusion assay o f the IgG, Fab and Fc reactions In fig. 3 (top) are represented the reactions of IgG and of its fragments with the two kinds o f antisera. In fig. 3 (bottom) the same experiment was performed with the antisera depleted of IgBC antibodies by absorbing on Sepharose IgBC. It can be seen that with respect to conventional serum (either horse or
chicken anti-rabbit serum) Fab and IgG show, as expected, a cross-reaction; Fab and Fc give a reaction of non-identity. (The Fc sample employed in this experiment was obtained on protein A-Sepharose, and it can be seen that it is contaminated by Fab). With respect to anti-IgBC serum, the reactions are quite different: Fab and IgG show a reaction of identity, this serum does not react with Fc (the slight precipitate is due to Fab contamination); it weakly precipitates IgBC. After the IgBC antibodies were removed, anti-IgBC serum no longer precipitated IgG or its fragments. By contrast adsorption with IgBC removed in conventional serum anti-Fab but not anti Fc antibodies.
3.5. Reaction with L and H chains For immunodiffusion assay, H chain was dissolved 149
i=a
Fc
I
/
/
\ A
anti_
Ig
(horse)
B
,,
,,
(chicken}
C
,,
IgBC
ly
D
"
Ig
,,
E
,,
Ig BC
IgGll
I
r-ab
E abs
Fig. 3. Double diffusion performed in Agar plates in NaC10.15 M, pH 7. IgG, Fab, Fc and IgBC were employed at the concentration of 0.5 mg/ml. A and B were conventional horse and chicken anti-rabbit serum and D was the serum B previously adsorbed with IgBC. C was chicken anti-lgBC serum and E the same serum adsorbed with IgBC.
in 8 M urea made in NaC1 0.15 M. Fig. 4 shows that again, as in the Fab reaction, precipitation of isolated H and L chains was quite different towards the two antisera. With IgBC antiserum L chain gives a strong precipitate; H and L chains are antigenically identical, L chain shows a strong line near the antiserum well which fuses with that of IgG and a second weaker line more exterior which also seems to rejoin the IgG precipitate. Thus, this antiserum probably recognizes K chain, which represents in rabbit about 90% of L chain stock, (strong precipitate) as well as ?~chain (weak line). With conventional serum the reactions are as expected, i.e. weak with L chain and strong with H chain. Moreover, L and H are antigenically different. IgG and H chain are antigenically identical because most of the antibodies of this serum are directed against the Fc fragment. 150
1 - anti2-"
Ig ( c h i c k e n ) -IgBC
"
Fig. 4. Double diffusion as in Fig. 3. IgG H and L chains were employed at 0.5 mg/ml.
4. Discussion The data of the present report clearly indicate that IgBC induces, in chickens, antibodies directed against IgM and IgG molecules. Apparently, anti-IgBC behaved as conventional anti-Ig sera; however, a fine analysis demonstrated that both were quite different. That the antigenic determinants (AgD) of the IgG molecule, which are recognized by anti-IgBC, are located in the Fab fragment exclusively was demonstrated not only by the failure to precipitate Fc but also by the antigenical identity of Fab and IgG against this antiserum. That was not a special feature of the animal employed to produce anti-IgBC, since chicken anti-normal Ig had the same characteristics as that induced in horse, that is, reacted mainly with the Fc piece in good agreement with the Porter's pioneer work [3]. The first evident conclusion is that anti-IgG antibodies in anti-IgBC sera had been induced by IgBC and not by an eventual IgG contamination. The finding that, by contrast with conventional antisera, anti-IgBC gave a good precipitin reaction with L chain, easily explains recognition of both IgM and IgG. It must in fact react with the other immunoglobulin classes since L chains are shared by all Ig. The fact that anti-IgBC precipitated H chain but not Fc indicates that the AgD common to IgBC and H chain are located in the Fd fragment. It must be concluded from these data that IgBC is contained in the Fd and the L chain structures. This immediately raises several problems concerning the way and the extent of participation to these structures as well as the origin of this substance. Ig molecules contain linked glucosides, but the bulk of them are located in the Fc fragment [6,7] thus the AgD shared by Fab and IgBC are probably supported by polypeptide sequences. The finding that absorption with IgBC removed in conventional serum the totality of the Fab precipitating antibodies suggests moreover that this substance is an important piece of these structures. As already pointed out, IgBC should be smaller than 13,700 daltons, thus smaller than the Fd and L chains. On the other hand, amino acid analyses have shown that it does not contain cysteine (Escribano, unpublished observation) and thus it cannot account for an entire domain. Since the AgD in Fab are probably spread across the two polypeptide chains, and
since there are several different antigenic determinants in these chains [8], IgBC probably contains a mixture of several peptides rather than a single unity. Concerning the origin of this substance, IgBC may arise from enzymatic degradation ofimmunoglobulins or alternatively be synthesized independently. The first hypothesis seems very improbable for the following reasons: (a) it is well known that Fab resists to enzyme attack; (b) IgBC was shown to be more abundant in the early rather than in the late immune response [2] or, in other words it is not proportional to the amount of antibody as should be expected in a degradation .event; and (c) Fab degradation fragments would not combine with IgG molecules as does IgBC. The second hypothesis implicates the existence of genes which can secrete independently and also be inserted into the complete DNA sequence coding for the entire L and H chains. Kabat et al. [9] have postulated the existence of minigenes for generating framework segments (FR), as well as complementary determining regions (CDR) [10]. Somatic assembly of those minigenes should generate the entire V-DNA encoding sequence [ 11 ]. This hypothesis is based on comparative studies of amino acid sequences of V domains in particular on the distribution of FR and CDR sets onto the immunoglobulin chains. At the moment, there is not, however, experimental support for the existence of minigenes except perhaps for that coding for the J piece [ 11 ,I 2]. Studies of DNA coding sequences seem, on the contrary, to refute this theory, since they have established that, although the sequences coding for V and C domains of mice light chain lie separated by a long base sequence [ 13], that coding for a complete domain is already contiguous in the embryo germ line [ 12,14] as well as in the adult myeloma cell [12,15]. If as our results suggest, IgBC is a product of independent synthesis on the one hand, and if on the other hand it contains several Ig fragments, it could be a good candidate to support the minigene hypothesis. It is thus very important to determine the extent of participation of IgBC to the Fab structure especially to see whether it is contained in the V or C domains. This search will be facilitated by the existence of genetic markers in rabbit C - L (b-allotypes) and V - H domains (a-allotypes). Structural studies are also needed to predict the exact nature of the IgBC pieces. Chemical studies can really be envis151
aged because IgBC is easily obtained, in relatively large amounts particularly from early antisera [1,2]. In connection with the problem already evoked that IgBC cannot account for an entire domain, it is worth emphasizing that it represents only the nondiffusible material of the Fil I fraction. It was indeed pointed out in the first paper consecrated to this fraction that it contained also diffusible peptides [ 1 ]. It will be interesting to determine the amino acid composition o f the diffusible material in order to see in particular if it contains the cysteine residues lacking in the non-diffusible IgBC. Finally, other chemical analysis already performed suggests that IgBC is a complex molecule made up not only of peptide but also of non-peptide material. The non-peptide moiety could be o f nucleotide nature as judged by the absorption band at 260 nm (Escribano, work in progress). Such a structure might explain the apparent paradox of IgBC being a (or several) polypeptide fragment(s) o f Fab and at same time having the property of combining with IgG. The non-peptide portion could be responsible for the IgG binding.
Acknowledgements The author thanks Professor P. Burtin for helpful discussion. Miss Annie Robbe, Mrs. Etiennette Daillan, Monique King and Mr. Noeum San are gratefully acknowledged for their excellent technical assistance.
152
References [1] Escribano, M. J. (1977)Comp. R. Acad. Sci. (Paris) s6rie D 284,693-695. [2] Escribano, M. J. (1978) Ann. Immunol. (Inst. Pasteur) 129C, 233-244. [3] Porter, R. R. (1959) Biochem. J. 73, 119-127. [4] Prahl, J. W. and Porter, R. R. (1968) Biochem. J. 107, 753-763. [5] Fleischman, J. B., Pain, R. H. and Porter, R. R. (1962) Arch. Biochem. Biophys., Suppl. 1, 174-180. [6] Nisonoff, A., Hopper, J. E. and Spring, S. B. (1975) in: The Antibody Molecule, Academic Press, New York. [7] Bergman, L. W. and Kuehl, W. M. (1977) Biochemistry 16, 4490-4497. [8] Mattlaews, J. B. and Jefferis, R. (1979) Mol. Immunol. (Formerly lmmunochemistry) 16,401-409. [9] Kabat, E. A., Wu, T. T. and Bilofsky, H. (1978) Proc. Nat. Acad. Sci. U.S.A. 75, 2429-2433. [10] Wu, T. T. and Kabat, E. A. (1970) J. Exp. Med. 132, 211-250. [ 11] Kabat, E. A., Wu, T. T. and Bilofsky, H. (1979) J. Exp. Med. 149, 1299-1319. [12] Bernard, O., Hozumi, N. and Tonegawa, S. (1978) Cell 15, 1133-1144. [13] Hozumi, N. and Tonegawa, S. (1976) Proc. Nat. Acad. Sci. U.S.A. 73, 3628-3632. [ 14] Tonegawa, S., Maxam, A. M., Tizard, R., Bernard, O. and Gilbert, W. (1978) Proc. Nat. Acad. Sci. U.S.A. 75, 1485 -1489. [15] Brack, Ch., Hirama, M., Lenhard-Schuller, R. and Tonegawa, S. (1978) Cell 15, 1-14.
© Elsevier/North-Holland Biomedical Press
AN IMMUNOGLOBULIN-BINDING COMPONENT FROM R A B B I T A N T I S E R A , B E A R I N G Fab ANTIGENIC DETERMINANTS M. J. ESCRIBANO Laboratoire d'Immunochirnie, I.R.S.C.; B.P. no. 8, 94800 Ville/uil; France
(Accepted August 31 1979)
1. Introduction A fraction of apparent low molecular weight called Fill, has regularly been found to be absorbed with the antibodies when rabbit immune sera were allowed to react with Sepharose-bound antigen. This fraction was present in antihapten as well as in antiprotein rabbit sera. It was separated from the antibodies on Sephadex G200, where it was eluted in third position after the IgM and IgG molecules. According to the elution volume on this resin, its molecular weight should be less than 13,700 [ 1,2]. FIII does not combine with the antigen but combines with the antibody [2]. This binding appears not to be related to the antibody activity since Fil I also combines with normal rabbit IgG; it seems, however, to be speciesspecific since among several species tested, namely rabbit, man, beef and chicken, only rabbit IgG showed a reaction with the rabbit Fii I fraction (M. J. Escribano, unpublished observation). This fraction referred here as an immunoglobulin-binding component (IgBC) was shown to be immunogenic in chickens. One antiserum, obtained after a single IgBC injection, precipitated this component in gel but did not, in particular, precipitate the immunoglobulins [ 1]. Thereafter, others chickens were immunized and the results were different since most of them did not give precipitating antibodies after the first injection. Immunization was then pursued and after two or more injections some sera had the particularity not only of precipitate IgBC but also the immunoglobulins IgG and IgM. In immunoelectrophoresis, the reaction with both Ig was indistinguishable from that observed with antisera obtained by immunization with normal rabbit serum (conven-
tional antiserum). These very peculiar antisera were then compared to conventional anti-lg sera and it was shown that both were functionally quite different. This was demonstrated by the following results: (1) anti-IgBC reacted with Fab but not with Fc fragment, whereas conventional antisera recognized, as expected, mainly Fc determinants;(2) absorption with IgBC removed in anti-IgBC sera all the anti-IgG antibodies. On the contrary, in conventional sera IgBC removed anti-Fab but not anti-Fc antibodies. These results indicate that the antigenic determinants which are recognized by anti-IgBC are located in the Fab fragment only and that moreover, those determinants are the same as the Fab determinants which are recognized by conventional sera. On the other hand, anti-IgBC precipitated H and L chains. IgBC behaves thus as one (or perhaps several) IgG fragments located in the Fab piece, in both L and Fd chains.
2. Material and methods IgBCpreparation. IgBC was obtained from either anti-DNP or anti-HGG (human gamma-globulin) rabbit sera as previously described [1,2]. Chicken anti-IgBC sera. Six chickens were immunized with IgBC from anti-HGG serum and 4 with IgBC from anti-DNP serum. In all instances 50-100/ag of IgBC were given subcutaneously in complete Freund's adjuvant two or more times. Each injection was spaced for 3 - 4 weeks. The animals were bled regularly 3 weeks after each injection. Rabbit IgG and IgM. Pure IgG and IgM were obtained by Sephadex G200 filtration of antibodies
147
obtained by irrmmno-adsorption from anti-HGG or anti-DNP sera of the early response (1 week after the first antigen injection) in order to obtain in particular IgM. Chicken anti-normal rabbit serum. Two chickens were immunized with normal rabbit serum, enriched of IgG and lgM prepared as above. Two injections of 0.5 ml enriched serum were done subcutaneously in complete Freund's adjuvant at a one month interval. The serum was collected 3 weeks after each injection. Horse anti-normal rabbit serum was obtained from the Institut Pasteur, Paris. lgBC imrmoloadsorbent. IgBC (a pool of several preparations) was coupled to BrCN activated Sepharose, (Pharmacia, Sweden). About 1 g resin containing 100/ag of covalently bound lgBC was prepared. To absorb anti-IgBC antibodies, the antisera were passed through the resin (which was packed in a small glass column), several times until all antibodies were removed. This was controlled by the measure of fixed antibodies which were eluted between each contact, with acid buffer. Finally the sera depleted o f anti-IgBC antibodies were concentrated in a Minicon Concentrator (Amicon, U.S.A.), until the initial volume was reconstituted. Preparation o f Fab and Fc fragments. Twenty mg of pure IgG prepared as above were digested by papain [3]. After dialysis against phosphate-buffered saline (P.B.S.) the digest was passed through a protein A-Sepharose (Pharmacia, Sweden) column. Protein A retains Fc efficiently and allows collection of Fab in pure grade. Acid elution of the bound material showed that it consisted not only of Fc but also of incompletely digested IgG and of a small quantity of Fab.
Pure Fc was obtained by the Porter's technic [3], (Pure Fc was a gift of Dr. G. Bordenave, Institut Pasteur Paris.) Preparation o f H and L chains. Pure IgG was reduced with dithiothreitol: (6 mg IgG dissolved in 0.4 ml Tris buffer pH 8.2 + 0.6 mg dithiothreitol, 1 h at room temperature), and alkylated with iodoacetic acid: (1.8 mg at -2°C 1 h) [4]. H and L chains were then separated on a Sephadex G75 column equilibrated with 1 M propionic acid [5]. Alternatively, L chain was obtained in a pure grade by adsorbing reduced IgG on protein A-Sepharose which retains H and unreduced IgG but not L chain.
3. Results 3.1. Antibodies in IgBC antisera About half of the immunized chickens formed precipitating anti-IgG antibodies from the second IgBC injection. Other sera did not give precipitin reactions but were able to agglutinate sheep red blood cells coated with IgG. Only precipitation reactions will be considered in this study. In general, no, or only very weak, precipitation was observed with the injected IgBC, the precipitation always being stronger with IgG than with IgBC. Finally the sera obtained by immunization with IgBC obtained from either DNP or HGG rabbit antisera had identical characteristics.
3.2. Reaction o f lgBC antisera with rabbit lgG and IgM The immunoelectrophoresis depicted in fig. 1, shows that anti-IgBC reacts with both Ig, and moreover
Fig. 1. lmmunoelectrophoresis of rabbit lgM and lgG developed with chicken anti-rabbit serum (conventional serum) and antiIgBC chicken serum. Electrophoresis was performed in Agargel (Oxoid, England) plates 10 cm length in barbital buffer pH 8.2 for 1 h under 100 V. IgM and IgG were employed at the concentration of 1 mg/ml. 148
-
~
~
÷
+
A
t Fab
B
D Q Fc A~_-
a n t i - l g (horse) ,, (chicken) - IgBC ,,
Fig. 2. Imunoelectrophoresis of isolated Fab and Fc fragments. The experimental conditions were the same as in Fig. 1. Fab and Fc were employed at the concentration of 1 mg/ml. A and B were developed with conventional antisera. A: horse anti-rabbit serum; B: chicken anti-rabbit serum. C and D were developed with two different anti-IgBC chicken sera.
the image is indistinguishable from that obtained with conventional antiserum (in this experiment conventional serum was chicken anti-rabbit serum).
3.3. Reaction with Fc and Fab Fig. 2 shows that whereas conventional antiserum (horse or chicken anti-rabbit serum), reacts with both Fab and Fc pieces, anti-IgBC precipitates only Fab. 3.4. Comparison in double diffusion assay o f the IgG, Fab and Fc reactions In fig. 3 (top) are represented the reactions of IgG and of its fragments with the two kinds o f antisera. In fig. 3 (bottom) the same experiment was performed with the antisera depleted of IgBC antibodies by absorbing on Sepharose IgBC. It can be seen that with respect to conventional serum (either horse or
chicken anti-rabbit serum) Fab and IgG show, as expected, a cross-reaction; Fab and Fc give a reaction of non-identity. (The Fc sample employed in this experiment was obtained on protein A-Sepharose, and it can be seen that it is contaminated by Fab). With respect to anti-IgBC serum, the reactions are quite different: Fab and IgG show a reaction of identity, this serum does not react with Fc (the slight precipitate is due to Fab contamination); it weakly precipitates IgBC. After the IgBC antibodies were removed, anti-IgBC serum no longer precipitated IgG or its fragments. By contrast adsorption with IgBC removed in conventional serum anti-Fab but not anti Fc antibodies.
3.5. Reaction with L and H chains For immunodiffusion assay, H chain was dissolved 149
i=a
Fc
I
/
/
\ A
anti_
Ig
(horse)
B
,,
,,
(chicken}
C
,,
IgBC
ly
D
"
Ig
,,
E
,,
Ig BC
IgGll
I
r-ab
E abs
Fig. 3. Double diffusion performed in Agar plates in NaC10.15 M, pH 7. IgG, Fab, Fc and IgBC were employed at the concentration of 0.5 mg/ml. A and B were conventional horse and chicken anti-rabbit serum and D was the serum B previously adsorbed with IgBC. C was chicken anti-lgBC serum and E the same serum adsorbed with IgBC.
in 8 M urea made in NaC1 0.15 M. Fig. 4 shows that again, as in the Fab reaction, precipitation of isolated H and L chains was quite different towards the two antisera. With IgBC antiserum L chain gives a strong precipitate; H and L chains are antigenically identical, L chain shows a strong line near the antiserum well which fuses with that of IgG and a second weaker line more exterior which also seems to rejoin the IgG precipitate. Thus, this antiserum probably recognizes K chain, which represents in rabbit about 90% of L chain stock, (strong precipitate) as well as ?~chain (weak line). With conventional serum the reactions are as expected, i.e. weak with L chain and strong with H chain. Moreover, L and H are antigenically different. IgG and H chain are antigenically identical because most of the antibodies of this serum are directed against the Fc fragment. 150
1 - anti2-"
Ig ( c h i c k e n ) -IgBC
"
Fig. 4. Double diffusion as in Fig. 3. IgG H and L chains were employed at 0.5 mg/ml.
4. Discussion The data of the present report clearly indicate that IgBC induces, in chickens, antibodies directed against IgM and IgG molecules. Apparently, anti-IgBC behaved as conventional anti-Ig sera; however, a fine analysis demonstrated that both were quite different. That the antigenic determinants (AgD) of the IgG molecule, which are recognized by anti-IgBC, are located in the Fab fragment exclusively was demonstrated not only by the failure to precipitate Fc but also by the antigenical identity of Fab and IgG against this antiserum. That was not a special feature of the animal employed to produce anti-IgBC, since chicken anti-normal Ig had the same characteristics as that induced in horse, that is, reacted mainly with the Fc piece in good agreement with the Porter's pioneer work [3]. The first evident conclusion is that anti-IgG antibodies in anti-IgBC sera had been induced by IgBC and not by an eventual IgG contamination. The finding that, by contrast with conventional antisera, anti-IgBC gave a good precipitin reaction with L chain, easily explains recognition of both IgM and IgG. It must in fact react with the other immunoglobulin classes since L chains are shared by all Ig. The fact that anti-IgBC precipitated H chain but not Fc indicates that the AgD common to IgBC and H chain are located in the Fd fragment. It must be concluded from these data that IgBC is contained in the Fd and the L chain structures. This immediately raises several problems concerning the way and the extent of participation to these structures as well as the origin of this substance. Ig molecules contain linked glucosides, but the bulk of them are located in the Fc fragment [6,7] thus the AgD shared by Fab and IgBC are probably supported by polypeptide sequences. The finding that absorption with IgBC removed in conventional serum the totality of the Fab precipitating antibodies suggests moreover that this substance is an important piece of these structures. As already pointed out, IgBC should be smaller than 13,700 daltons, thus smaller than the Fd and L chains. On the other hand, amino acid analyses have shown that it does not contain cysteine (Escribano, unpublished observation) and thus it cannot account for an entire domain. Since the AgD in Fab are probably spread across the two polypeptide chains, and
since there are several different antigenic determinants in these chains [8], IgBC probably contains a mixture of several peptides rather than a single unity. Concerning the origin of this substance, IgBC may arise from enzymatic degradation ofimmunoglobulins or alternatively be synthesized independently. The first hypothesis seems very improbable for the following reasons: (a) it is well known that Fab resists to enzyme attack; (b) IgBC was shown to be more abundant in the early rather than in the late immune response [2] or, in other words it is not proportional to the amount of antibody as should be expected in a degradation .event; and (c) Fab degradation fragments would not combine with IgG molecules as does IgBC. The second hypothesis implicates the existence of genes which can secrete independently and also be inserted into the complete DNA sequence coding for the entire L and H chains. Kabat et al. [9] have postulated the existence of minigenes for generating framework segments (FR), as well as complementary determining regions (CDR) [10]. Somatic assembly of those minigenes should generate the entire V-DNA encoding sequence [ 11 ]. This hypothesis is based on comparative studies of amino acid sequences of V domains in particular on the distribution of FR and CDR sets onto the immunoglobulin chains. At the moment, there is not, however, experimental support for the existence of minigenes except perhaps for that coding for the J piece [ 11 ,I 2]. Studies of DNA coding sequences seem, on the contrary, to refute this theory, since they have established that, although the sequences coding for V and C domains of mice light chain lie separated by a long base sequence [ 13], that coding for a complete domain is already contiguous in the embryo germ line [ 12,14] as well as in the adult myeloma cell [12,15]. If as our results suggest, IgBC is a product of independent synthesis on the one hand, and if on the other hand it contains several Ig fragments, it could be a good candidate to support the minigene hypothesis. It is thus very important to determine the extent of participation of IgBC to the Fab structure especially to see whether it is contained in the V or C domains. This search will be facilitated by the existence of genetic markers in rabbit C - L (b-allotypes) and V - H domains (a-allotypes). Structural studies are also needed to predict the exact nature of the IgBC pieces. Chemical studies can really be envis151
aged because IgBC is easily obtained, in relatively large amounts particularly from early antisera [1,2]. In connection with the problem already evoked that IgBC cannot account for an entire domain, it is worth emphasizing that it represents only the nondiffusible material of the Fil I fraction. It was indeed pointed out in the first paper consecrated to this fraction that it contained also diffusible peptides [ 1 ]. It will be interesting to determine the amino acid composition o f the diffusible material in order to see in particular if it contains the cysteine residues lacking in the non-diffusible IgBC. Finally, other chemical analysis already performed suggests that IgBC is a complex molecule made up not only of peptide but also of non-peptide material. The non-peptide moiety could be o f nucleotide nature as judged by the absorption band at 260 nm (Escribano, work in progress). Such a structure might explain the apparent paradox of IgBC being a (or several) polypeptide fragment(s) o f Fab and at same time having the property of combining with IgG. The non-peptide portion could be responsible for the IgG binding.
Acknowledgements The author thanks Professor P. Burtin for helpful discussion. Miss Annie Robbe, Mrs. Etiennette Daillan, Monique King and Mr. Noeum San are gratefully acknowledged for their excellent technical assistance.
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