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Antibodies against protein antigenic sites that are identical in the homologous protein of the immunized animal Autoreactivity in rabbits of antibodies to spermwhale myoglobin
Biochimica et Biophysica Acta, 4 9 4 ( 1 9 7 7 ) 2 7 7 - - 2 8 2 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA R e p o r t BBA 31231
ANTIBODIES AGAINST P R O T E I N ANTIGENIC SITES THAT A R E IDENTICAL IN THE H O M O L O G O U S PROTEIN OF THE IMMUNIZED ANIMAL A U T O R E A C T I V I T Y IN RABBITS OF ANTIBODIES TO SPERMWHALE MYOGLOBIN
A. L A T I F K A Z I M a n d M.Z. A T A S S I *
Department of Immunology, Mayo Medical School, Rochester, Minn. 55901 and Department of Biochemistry, University of Minnesota, Minneapolis, Minn. 55455 (U.S.A.) (Received June 6th, 1977)
Summary Sequence comparisons between the antigenic sites of sperm-whale myogiobin and the corresponding regions in rabbit myoglobin indicate that rabbits make antibodies to regions of the sperm-whale myoglobin molecule which are identical to the corresponding regions in rabbit myoglobin. Rabbit myoglobin did not precipitate with antisera to sperm-whale myoglobin. However, it exhibited an extensive cross-reaction as demonstrated by its ability to inhibit the precipitin reaction of sperm-whale myoglobin, and on an immunoadsorbent, b o u n d a large a m o u n t of antibodies to sperm-whale myoglobin.
Previous work from this laboratory [ 1] had completely elucidated the antigenic structure of sperm-whale myoglobin (Mb). The antigenic reactive sites of sperm-whale Mb have been shown to consist of continuous portions of its primary sequence and are located in conformationally distinct portions of the molecule [ 1]. The conservation of overall three-dimensional structure among globin chains, and occasionally extensive homologies in their amino acid sequences p r o m p t e d us to examine whether, in responding to sperm-whale Mb, the host animal will make antibodies to regions of the sperm-whale Mb molecule which are similar or identical to the corresponding *To w h o m c o r r e s p o n d e n c e should be a d d r e s s e d at D e p a r t m e n t o f I m m u n o l o g y , M a y o MedicaJ S c h o o l , R o c h e s t e r ~ Minn. 55901, U.S.A. A b b r e v i a t i o n s : Mb, myoglobin; SW Mb and R Mb refer to sperm-whale myoglobin and rabbit myoglobin~ respectively ~SDS, sodium dodecyl sulfate.
278
regions in the animal's own Mb. The recent availability of the primary structure of rabbit Mb [2] afforded us the opportunity for such studies. A comparison of the primary structure of sperm-whale Mb with that of Mb from the rabbit, an animal in which antisera to sperm-whale Mb has been made, shows that both are of identical chain length (153 amino acids), and differ in sequence at 22 amino acid locations. Of the 22 residues by which sperm-whale and rabbit Mb differ, only 5 of these fall within the boundaries of the antigenic sites recognized by rabbit antibodies to spermwhale Mb. In Fig. 1 the primary sequences of the five antigenic sites of sperm-whale Mb are shown together with the corresponding regions from rabbit Mb. The amino acid replacements which occur in the corresponding rabbit Mb sequences are : Ala is -+ Gly and Va121 ---> Leu in region 15--22, Arg 1'8 -~ Lys in region 113--119, and Lys 14s ~ Gln and Tyr 'sl ~ Phe in region 145--151. Two regions, 56--62 and 94--99, are identical in both myoglobins. The close similarity of the two myogiobins in these regions was surprising to us in view of the belief that an animal would not make antibodies to regions of a protein antigen which were identical to corresponding regions in the animal's own protein [3]. These similarities also suggested that barring any drastic conformational differences rabbit antibodies to spermwhale Mb could also react with rabbit Mb. Since both rabbit and goat antibodies to sperm-whale Mb recognize identical antigenic sites {shown in Fig. 1) on the sperm-whale Mb molecule [4--8], goat antibodies to sperm-whale Mb may also be expected to react with rabbit Mb. Rabbit Mb was prepared by chromatography on CM-cellulose as prey-
SITE I of
Sperm-whale Mb Rabbit Mb
15 ~ ~Gly[
Lys Lys
Val Val
Glu Glu
Ala Ala
Asp Asp
SITE II of
Sperm-whale Mb Rabbit Mb
56 Lys Lys
Ala Ala
Set Ser
Glu Glu
Asp Asp
Leu Leu
SITE II1 of Sperm-whale Mb Rabbit Mb
94 Ala Ala
Thr Thr
Lys Lys
His His
Lys Lys
99 lle Ile
SITE IV of
Sperm-whale Mb Rabbit Mb
113 His His
Val Val
Leu Leu
His His
Ser Ser
SITE V of
Sperm-whale Mb Rabbit Mb
[45 ~-~ ~
Tyr Tyr
Lys Lys
Glu Glu
Leu Leu
22 ~-~ AIm ~Leu~ Ala
62 Lys Lys
119 ~] His [Lys[ His
Gly Gly
151 ~
Fig. 1. A d i a g r a m s h o w i n g t h e primary structures o f t h e five a n t i g e n i c sites o f s p e r m - w h a l e M b a n d t h e c o r r e s p o n d i n g h o m o l o g o u s r e g i o n s o f r a b b i t M b . T h e s e q u e n c e s s h o w n o c c u p y i d e n t i c a l p o s i t i o n s in t h e r e s p e c t i v e p r o t e i n c h a i n s . I d e n t i c a l p o s i t i o n s h a v i n g d i f f e r e n t a m i n o acids in t h e t w o c h a i n s are indicated by blocks.
279 iously described for Mb from other species [ 9 ] , and was shown by disc gel electrophoresis in the absence and presence of SDS to be homogeneous with a molecular weight of 17 000--18 000. Amino acid analysis yielded the expected composition. Antisera to sperm-whale Mb were raised in both rabbits and goats by immunization schedules already described [ 1 0 ] . Quantitative precipitin and inhibition reactions were done as previously described [ 11 ]. Rabbit and sperm-whale myoglobins were coupled to cyanogen bromide activated Sepharose-4B as previously described for sperm-whale Mb [ 1 2 ] . Immunoadsorption studies were performed essentially as described [ 12] except that the antibody b o u n d after incubation of the antisera with the adsorbents and subsequent washing with 0.15 M NaC1/0.01 M sodium phosphate, pH 7.0, to remove nonspecifically adsorbed protein, were eluted with 5 M guanidine, pH 8.5. The eluted antibody was quantitated by Lowry protein determinations after dialysis of the eluted fractions against 0.0175 M phosphate buffer, pH 7.0. A control adsorbent consisting of hen egg-white lysozyme conjugated to Sepharose 4-B was incubated with these antisera and eluted under identical conditions. Protein b o u n d to this lysozyme adsorbent was considered to be non-specifically adsorbed, and the protein eluted from the sperm-whale Mb and rabbit Mb adsorbents were corrected for this nonspecific background adsorption. As judged by double diffusion in agar and quantitative precipitin methods, rabbit Mb did n o t precipitate with either rabbit or goat antibodies to sperm-whale Mb. However, rabbit Mb was found to effectively inhibit the quantitative precipitin reaction of sperm-whale Mb with both rabbit and goat antibodies to sperm-whale Mb. The inhibition curve obtained with rabbit antiserum 77, to sperm-whale Mb is shown in Fig. 2. This antiserum may be considered to be representative of rabbit antisera to spermwhale Mb. During the course of the delineation of the antigenic structure of sperm-whale Mb a number of different rabbit antisera were shown to have identical specificities with respect to the antigenic sites on spermwhale Mb which they recognized, differing only in the relative amounts of site-specific antibodies [ 1]. Table I summarizes the results of the inhibi75
5O Z
I
•
/
I
I
I
i
_~ 25
L 0
I 50 RMb/SWMb
I MOLAR
I 100 RATIO
~ 150
F i g . 2. Q u a n t i t a t i v e i n h i b i t i o n b y r a b b i t M b o f t h e p r e c i p i t i n r e a c t i o n o f s p e r m - w h a l e Mb w i t h o n e r a b b i t a n t i s e r u m ( 7 7 ) . E a c h p o i n t r e p r e s e n t s t h e average o f t h r e e d e t e r m i n a t i o n s . F o r o t h e r i n h i b i t i o n r e s u l t s , see T a b l e I.
280 TABLE I I N H I B I T O R Y A C T I V I T I E S O F R A B B I T Mb I N T H E S P E R M - W H A L E M b - A N T I SW Mb P R E C I P ITIN REACTION A n t i s e r a w e r e r a i s e d a g a i n s t s p e ~ n - w h a l e Mb in r a b b i t ( 7 7 ) a n d g o a t ( G 3 , G 4 ) as r e f e r e n c e d in t h e t e x t . I n h i b i t i o n v a l u e s are e x p r e s s e d as m a x i m u m p e r c e n t i n h i b i t i o n b y r a b b i t Mb o f the p r e c i p i t i n r e a c t i o n o f s p e r m - w h a l e Mb w i t h t h e a n t i s e r a s h o w n . E a c h v a l u e is a n a v e r a g e of a t least t h r e e d e t e r m i n a t i o n s w h i c h v a r i e d ± 1.5% o r less. Antiserum
M a x i m u m i n h i b i t i o n (%)
M o l a r r a t i o ( R M b / S W Mb) at h a l f m a x . i n h i b i t i o n *
77 G3 G4
72 76 90
19 12 3
* T h e s e v a l u e s r e p r e s e n t t h e r a b b i t M b / s p e r m - w h a l e Mb m o l a r r a t i o s a t 50% m a x i m u m i n h i b i t i o n a n d are d e r i v e d f r o m i n h i b i t i o n c u r v e s similar t o t h a t s h o w n in Fig. 2.
tion reactions obtained with antiserum 77, as well as with two goat antisera to sperm-whale Mb, G3 and G4 (which, as previously mentioned, recognized the same antigenic sites on sperm-whale Mb as the rabbit antisera). The large inhibitions obtained with these antisera indicate that rabbit Mb interacts effectively with both rabbit and goat antisera to sperm-whale Mb. As judged by the lower molar excess required for half m a x i m u m inhibition, antiserum G4 exhibits a higher affinity for rabbit Mb, than either antisera G3 or 77, while the affinities of G3 and 77 are of comparable magnitude. A further test of the reactivity of rabbit Mb with antisera to spermwhale Mb was performed by examining the ability of an immunoadsorbent of rabbit Mb to bind antibodies from antisera to sperm-whale Mb. Table II summarizes the results with two rabbit (77 and 80) and one goat (G4) antisera. The values in Table II have been corrected for non-specifically adsorbed protein bound to Lysozyme-Sepharose under identical conditions. These corrections were no more than 5--10% of the total bound to the SW Mb-Sepharose adsorbent. From the values shown in Table II, it is evident that a substantial portion of the antibodies to sperm-whale Mb could be adsorbed by the rabbit Mb adsorbent. Furthermore, it should be mentioned that fractions of the antibodies that could not be bound on the rabbit Mb adsorbent were recovered quantitatively by subsequent passage on an immunoadsorbent of sperm-whale Mb. The total amounts of anti-
TABLE II SUMMARY OF IMMU/qOADSORBENT STUDIES A n t i s e r a axe t h e s a m e as t h o s e r e f e r r e d t o in T a b l e I e x c e p t N o . 80 w h i c h is a r a b b i t a n t i s e r u m to s p e r m - w h a l e Mb. Values are e x p r e s s e d as p e r c e n t o f t h e t o t a l a n t i b o d i e s a d s o r b e d b y a r a b b i t Mb a d s o r b e n t r e l a t i v e t o t h o s e b o u n d b y a s p e r m - w h a l e Mb a d s o r b e n t as 1 0 0 % . Antiserum
A n t i SW Mb a d s o r b e d b y SW M b - S e p h a r o s e (%)*
A n t i SW Mb a d s o r b e d b y R M b - S e p h a r o s e (%)*
77 80 G4
100 100 100
42.9 65.8 49.6
* T h e s e values h a v e b e e n c o r r e c t e d f o r n o n - s p e c i f i c a l l y a d s o r b e d p r o t e i n r e t a i n e d o n a L y s o z y m e Sepha~ose a d s o r b e n t , as e x p l a i n e d in the t e x t .
281 bodies which could be recovered by elution from serial passage through a rabbit Mb followed by a sperm-whale Mb adsorbent column averaged 90--95% of the total antibodies adsorbed by passage of the antisera over a sperm-whale Mb adsorbent alone. The extensive ability of rabbit Mb to interact with antibodies to spermwhale Mb leads to a consideration of the sites through which the rabbit Mb may interact with these antisera. Regions 56--62 and 94--99 are identical in both myoglobins {Fig. 1), and unless their conformations have been altered through amino acid replacements not located in these regions, these two sites would be expected to react completely. With respect to regions 15--22 and 113--119, our previous studies have shown that the binding of reactive regions to antibodies is primarily effected through polar interactions, with non-polar amino acids providing more of a stabilizing role through hydrophobic interactions [ 1 3 ] . Based on these considerations and on the conservative character of the amino acid substitutions in region 15--22 (Ala Is -~ Gly, Va121 -~ Leu) and 113--119 (Arg ~8 -* Lys), these two regions in rabbit Mb would n o t be expected to be completely unreactive. However, it cannot be excluded that subtle conformational changes in these regions directed by these replacements as well as by replacements elsewhere in the rabbit Mb molecule, could diminish the affinity of these sites for their respective antibodies. Although Lys-145 is included in the reactive region 145--151, it plays only a marginal role with some antisera and, of those studied here, is required only for the reaction of the free peptide 145--151 with antiserum G4, b u t is not part of the reactive region in intact, native spermwhale Mb [4]. Therefore, the replacement of Lys-145 by glutamine in rabbit Mb should be w i t h o u t consequence to the reactivity of this region in native rabbit Mb. However, Tyr-151 has been shown to be absolutely essential for the reactivity of this region, and its replacement by phenylalanine completely obliterates its reactivity [ 1 4 ] . Rabbit Mb is, therefore, not expected to react with antibodies to sperm-whale Mb through region 145--151. The expected diminished reactivities for region 15--22 and 113--119 together with the complete unreactivity of region 145--151 would account for the lack of total cross-reactivity of these t w o myoglobins observed by the precipitin inhibition and immunoadsorption results presented above. The foregoing studies clearly indicate that rabbits respond to immunization with sperm-whale Mb by producing antibodies that are directed against regions of the sperm-whale Mb molecule which are both identical {regions 5 6 - 6 2 and 94--99) and closely similar (regions 15--22 and 113-119) to the corresponding sequences in the rabbit's own Mb. Furthermore, these antibodies will extensively crossreact with rabbit Mb through these equivalent regions. As mentioned above both rabbits and goats make antibodies directed against regions of the sperm-whale Mb molecule which are identical in their structures and locations [4--8]. Pertinent, in this regard, is that hen egg-white lysozyme, the second protein whose antigenic structure has been completely determined [ 15--17 ], is also recognized through identical antigenic sites by both rabbit and goat antisera. In a pilot experiment we have also observed that goat Mb is extremely effective in
282 inhibiting the precipitin reaction of sperm-whale Mb with goat antisera to sperm-whale Mb (unpublished results). Although the sequence of goat Mb is not available for comparison at the present time, we predict that similar identities exist between the reactive regions of sperm-whale Mb and the corresponding regions of goat Mb. It is apparent, therefore, that the antigenic sites of sperm-whale Mb are characteristic for sperm-whale Mb, at least with respect to rabbits and goats, and are independent of any sequence identities which m a y occur between sperm-whale Mb and rabbit Mb or sperm-whale Mb and goat Mb. Accordingly, approaches which attempt to localize antigenic sites of proteins by comparing the primary structure of the antigen with that of the homologous protein in the immunized animal [3] will most likely have to be re-evaluated. It is worthwhile to note here that while Mb is an intramuscular protein thought to be sheltered from exposure to the immune system, its presence in normal serum has been demonstrated [18]. The induction of autoreactive antibodies to "sequestered" antigens by immunization with a cross-reacting antigen is not unprecedented (for review see ref. 19). However, the lack of knowledge concerning the molecular structures and locations of protein antigenic sites has lead to uncertainty regarding the precise regions of the proteins through which these cross-reactions are effected. This is the first system to be described in which the structures of the antigenic sites of the cross-reacting protein are completely known, and provides an excellent model for studying some of the molecular aspects of immunologic tolerance to auto-antigens and its termination. The technical assistance of Mr. Duane Richardson is gratefully acknowledged The work was supported by a grant (AM 18920) from the Institute of Arthritis and Metabolic Diseases, National Institutes of Health, U.S. Public Health Service. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Atassi, M.Z. (1975) I m m u n o c h e m l s t r y 12, 423--438 Romero-Herrera, A.E., Lehmann, H. and Castillo, O. (1976) Biochim. Biophys. Acta 439, 51--54 ReichHn, M. (1972) J. Mol. Biol. 6 4 , 4 8 5 - - 4 9 6 Koketsu, J. and Atassi, M.Z. (1973) Biochim. Biophys. Acta 328, 289--302 Koketsu, J. and Atassi, M.Z. (1974) I m m u n o c h e m i s t r y 11, 1--8 Koketsu, J. and Atassi, M.Z. (1974) Biochim. Biophys. Acta 342, 21--29 Pal, R.-C. and Atassi, M.Z. (1975) I m m u n o c h e m i s t r y 12, 285--290 Atassi, M.Z. and Pal, R.-C. (1975) I m m u n o c h e m i s t r y 12, 735--740 Atassi, M.Z. (1970) Biochim. Biophys. Acta 221,612---622 Atassi, M.Z. (1967) Biochem. J. 102, 478--487 Atassi, M.Z. and Saplin, B.J. (1968) Biochemistry 7 , 6 8 8 - - 6 9 8 Atassi, M.Z, and Koketsu, J. (1975) I m m u n o c h e m i s t r y 12, 741--744 Atassl, M.Z. (1972) in Specific Receptors of Antibodies, Antigens and Cells, 3rd Int e rna t i ona l Convocation of I m m u n o l o g y , June 12--15 (Pressman, D., Tomasi, T.B., Grossberg, A.L. and Rose, N.R., eds.), pp. 118--136, Karger, Base| Atassi, M.Z. and Saplln, B.J. (1971) Biochemistry 10, 4 7 4 0 - - 4 7 4 7 Atassi, M.Z. , Koketsu, J. and Habeeb, A.F.S.A. (1976) Biochim. Biophys. Acta 420, 358--375 Atass|, M.Z., Lee, C.-L. and Pal, R,-C. (1976) Biochim. Biophys. Acta 427, 745--751 Lee, C.-L. and Atassl, M.Z. (1976) Biochem. J. 157, 89---93 Reichlin, M., Visco, J.P. and Klocke, F.J. (1976) Cl~n. Res. 2 4 , 4 2 1 (Abstr.) Bergst~and, H. (1977) in I m m u n o c h e m i s t r y of Proteins (Atassi0 M.Z., ed.), Vol. 1, pp. 315-370, Plenum, New York
BBA R e p o r t BBA 31231
ANTIBODIES AGAINST P R O T E I N ANTIGENIC SITES THAT A R E IDENTICAL IN THE H O M O L O G O U S PROTEIN OF THE IMMUNIZED ANIMAL A U T O R E A C T I V I T Y IN RABBITS OF ANTIBODIES TO SPERMWHALE MYOGLOBIN
A. L A T I F K A Z I M a n d M.Z. A T A S S I *
Department of Immunology, Mayo Medical School, Rochester, Minn. 55901 and Department of Biochemistry, University of Minnesota, Minneapolis, Minn. 55455 (U.S.A.) (Received June 6th, 1977)
Summary Sequence comparisons between the antigenic sites of sperm-whale myogiobin and the corresponding regions in rabbit myoglobin indicate that rabbits make antibodies to regions of the sperm-whale myoglobin molecule which are identical to the corresponding regions in rabbit myoglobin. Rabbit myoglobin did not precipitate with antisera to sperm-whale myoglobin. However, it exhibited an extensive cross-reaction as demonstrated by its ability to inhibit the precipitin reaction of sperm-whale myoglobin, and on an immunoadsorbent, b o u n d a large a m o u n t of antibodies to sperm-whale myoglobin.
Previous work from this laboratory [ 1] had completely elucidated the antigenic structure of sperm-whale myoglobin (Mb). The antigenic reactive sites of sperm-whale Mb have been shown to consist of continuous portions of its primary sequence and are located in conformationally distinct portions of the molecule [ 1]. The conservation of overall three-dimensional structure among globin chains, and occasionally extensive homologies in their amino acid sequences p r o m p t e d us to examine whether, in responding to sperm-whale Mb, the host animal will make antibodies to regions of the sperm-whale Mb molecule which are similar or identical to the corresponding *To w h o m c o r r e s p o n d e n c e should be a d d r e s s e d at D e p a r t m e n t o f I m m u n o l o g y , M a y o MedicaJ S c h o o l , R o c h e s t e r ~ Minn. 55901, U.S.A. A b b r e v i a t i o n s : Mb, myoglobin; SW Mb and R Mb refer to sperm-whale myoglobin and rabbit myoglobin~ respectively ~SDS, sodium dodecyl sulfate.
278
regions in the animal's own Mb. The recent availability of the primary structure of rabbit Mb [2] afforded us the opportunity for such studies. A comparison of the primary structure of sperm-whale Mb with that of Mb from the rabbit, an animal in which antisera to sperm-whale Mb has been made, shows that both are of identical chain length (153 amino acids), and differ in sequence at 22 amino acid locations. Of the 22 residues by which sperm-whale and rabbit Mb differ, only 5 of these fall within the boundaries of the antigenic sites recognized by rabbit antibodies to spermwhale Mb. In Fig. 1 the primary sequences of the five antigenic sites of sperm-whale Mb are shown together with the corresponding regions from rabbit Mb. The amino acid replacements which occur in the corresponding rabbit Mb sequences are : Ala is -+ Gly and Va121 ---> Leu in region 15--22, Arg 1'8 -~ Lys in region 113--119, and Lys 14s ~ Gln and Tyr 'sl ~ Phe in region 145--151. Two regions, 56--62 and 94--99, are identical in both myoglobins. The close similarity of the two myogiobins in these regions was surprising to us in view of the belief that an animal would not make antibodies to regions of a protein antigen which were identical to corresponding regions in the animal's own protein [3]. These similarities also suggested that barring any drastic conformational differences rabbit antibodies to spermwhale Mb could also react with rabbit Mb. Since both rabbit and goat antibodies to sperm-whale Mb recognize identical antigenic sites {shown in Fig. 1) on the sperm-whale Mb molecule [4--8], goat antibodies to sperm-whale Mb may also be expected to react with rabbit Mb. Rabbit Mb was prepared by chromatography on CM-cellulose as prey-
SITE I of
Sperm-whale Mb Rabbit Mb
15 ~ ~Gly[
Lys Lys
Val Val
Glu Glu
Ala Ala
Asp Asp
SITE II of
Sperm-whale Mb Rabbit Mb
56 Lys Lys
Ala Ala
Set Ser
Glu Glu
Asp Asp
Leu Leu
SITE II1 of Sperm-whale Mb Rabbit Mb
94 Ala Ala
Thr Thr
Lys Lys
His His
Lys Lys
99 lle Ile
SITE IV of
Sperm-whale Mb Rabbit Mb
113 His His
Val Val
Leu Leu
His His
Ser Ser
SITE V of
Sperm-whale Mb Rabbit Mb
[45 ~-~ ~
Tyr Tyr
Lys Lys
Glu Glu
Leu Leu
22 ~-~ AIm ~Leu~ Ala
62 Lys Lys
119 ~] His [Lys[ His
Gly Gly
151 ~
Fig. 1. A d i a g r a m s h o w i n g t h e primary structures o f t h e five a n t i g e n i c sites o f s p e r m - w h a l e M b a n d t h e c o r r e s p o n d i n g h o m o l o g o u s r e g i o n s o f r a b b i t M b . T h e s e q u e n c e s s h o w n o c c u p y i d e n t i c a l p o s i t i o n s in t h e r e s p e c t i v e p r o t e i n c h a i n s . I d e n t i c a l p o s i t i o n s h a v i n g d i f f e r e n t a m i n o acids in t h e t w o c h a i n s are indicated by blocks.
279 iously described for Mb from other species [ 9 ] , and was shown by disc gel electrophoresis in the absence and presence of SDS to be homogeneous with a molecular weight of 17 000--18 000. Amino acid analysis yielded the expected composition. Antisera to sperm-whale Mb were raised in both rabbits and goats by immunization schedules already described [ 1 0 ] . Quantitative precipitin and inhibition reactions were done as previously described [ 11 ]. Rabbit and sperm-whale myoglobins were coupled to cyanogen bromide activated Sepharose-4B as previously described for sperm-whale Mb [ 1 2 ] . Immunoadsorption studies were performed essentially as described [ 12] except that the antibody b o u n d after incubation of the antisera with the adsorbents and subsequent washing with 0.15 M NaC1/0.01 M sodium phosphate, pH 7.0, to remove nonspecifically adsorbed protein, were eluted with 5 M guanidine, pH 8.5. The eluted antibody was quantitated by Lowry protein determinations after dialysis of the eluted fractions against 0.0175 M phosphate buffer, pH 7.0. A control adsorbent consisting of hen egg-white lysozyme conjugated to Sepharose 4-B was incubated with these antisera and eluted under identical conditions. Protein b o u n d to this lysozyme adsorbent was considered to be non-specifically adsorbed, and the protein eluted from the sperm-whale Mb and rabbit Mb adsorbents were corrected for this nonspecific background adsorption. As judged by double diffusion in agar and quantitative precipitin methods, rabbit Mb did n o t precipitate with either rabbit or goat antibodies to sperm-whale Mb. However, rabbit Mb was found to effectively inhibit the quantitative precipitin reaction of sperm-whale Mb with both rabbit and goat antibodies to sperm-whale Mb. The inhibition curve obtained with rabbit antiserum 77, to sperm-whale Mb is shown in Fig. 2. This antiserum may be considered to be representative of rabbit antisera to spermwhale Mb. During the course of the delineation of the antigenic structure of sperm-whale Mb a number of different rabbit antisera were shown to have identical specificities with respect to the antigenic sites on spermwhale Mb which they recognized, differing only in the relative amounts of site-specific antibodies [ 1]. Table I summarizes the results of the inhibi75
5O Z
I
•
/
I
I
I
i
_~ 25
L 0
I 50 RMb/SWMb
I MOLAR
I 100 RATIO
~ 150
F i g . 2. Q u a n t i t a t i v e i n h i b i t i o n b y r a b b i t M b o f t h e p r e c i p i t i n r e a c t i o n o f s p e r m - w h a l e Mb w i t h o n e r a b b i t a n t i s e r u m ( 7 7 ) . E a c h p o i n t r e p r e s e n t s t h e average o f t h r e e d e t e r m i n a t i o n s . F o r o t h e r i n h i b i t i o n r e s u l t s , see T a b l e I.
280 TABLE I I N H I B I T O R Y A C T I V I T I E S O F R A B B I T Mb I N T H E S P E R M - W H A L E M b - A N T I SW Mb P R E C I P ITIN REACTION A n t i s e r a w e r e r a i s e d a g a i n s t s p e ~ n - w h a l e Mb in r a b b i t ( 7 7 ) a n d g o a t ( G 3 , G 4 ) as r e f e r e n c e d in t h e t e x t . I n h i b i t i o n v a l u e s are e x p r e s s e d as m a x i m u m p e r c e n t i n h i b i t i o n b y r a b b i t Mb o f the p r e c i p i t i n r e a c t i o n o f s p e r m - w h a l e Mb w i t h t h e a n t i s e r a s h o w n . E a c h v a l u e is a n a v e r a g e of a t least t h r e e d e t e r m i n a t i o n s w h i c h v a r i e d ± 1.5% o r less. Antiserum
M a x i m u m i n h i b i t i o n (%)
M o l a r r a t i o ( R M b / S W Mb) at h a l f m a x . i n h i b i t i o n *
77 G3 G4
72 76 90
19 12 3
* T h e s e v a l u e s r e p r e s e n t t h e r a b b i t M b / s p e r m - w h a l e Mb m o l a r r a t i o s a t 50% m a x i m u m i n h i b i t i o n a n d are d e r i v e d f r o m i n h i b i t i o n c u r v e s similar t o t h a t s h o w n in Fig. 2.
tion reactions obtained with antiserum 77, as well as with two goat antisera to sperm-whale Mb, G3 and G4 (which, as previously mentioned, recognized the same antigenic sites on sperm-whale Mb as the rabbit antisera). The large inhibitions obtained with these antisera indicate that rabbit Mb interacts effectively with both rabbit and goat antisera to sperm-whale Mb. As judged by the lower molar excess required for half m a x i m u m inhibition, antiserum G4 exhibits a higher affinity for rabbit Mb, than either antisera G3 or 77, while the affinities of G3 and 77 are of comparable magnitude. A further test of the reactivity of rabbit Mb with antisera to spermwhale Mb was performed by examining the ability of an immunoadsorbent of rabbit Mb to bind antibodies from antisera to sperm-whale Mb. Table II summarizes the results with two rabbit (77 and 80) and one goat (G4) antisera. The values in Table II have been corrected for non-specifically adsorbed protein bound to Lysozyme-Sepharose under identical conditions. These corrections were no more than 5--10% of the total bound to the SW Mb-Sepharose adsorbent. From the values shown in Table II, it is evident that a substantial portion of the antibodies to sperm-whale Mb could be adsorbed by the rabbit Mb adsorbent. Furthermore, it should be mentioned that fractions of the antibodies that could not be bound on the rabbit Mb adsorbent were recovered quantitatively by subsequent passage on an immunoadsorbent of sperm-whale Mb. The total amounts of anti-
TABLE II SUMMARY OF IMMU/qOADSORBENT STUDIES A n t i s e r a axe t h e s a m e as t h o s e r e f e r r e d t o in T a b l e I e x c e p t N o . 80 w h i c h is a r a b b i t a n t i s e r u m to s p e r m - w h a l e Mb. Values are e x p r e s s e d as p e r c e n t o f t h e t o t a l a n t i b o d i e s a d s o r b e d b y a r a b b i t Mb a d s o r b e n t r e l a t i v e t o t h o s e b o u n d b y a s p e r m - w h a l e Mb a d s o r b e n t as 1 0 0 % . Antiserum
A n t i SW Mb a d s o r b e d b y SW M b - S e p h a r o s e (%)*
A n t i SW Mb a d s o r b e d b y R M b - S e p h a r o s e (%)*
77 80 G4
100 100 100
42.9 65.8 49.6
* T h e s e values h a v e b e e n c o r r e c t e d f o r n o n - s p e c i f i c a l l y a d s o r b e d p r o t e i n r e t a i n e d o n a L y s o z y m e Sepha~ose a d s o r b e n t , as e x p l a i n e d in the t e x t .
281 bodies which could be recovered by elution from serial passage through a rabbit Mb followed by a sperm-whale Mb adsorbent column averaged 90--95% of the total antibodies adsorbed by passage of the antisera over a sperm-whale Mb adsorbent alone. The extensive ability of rabbit Mb to interact with antibodies to spermwhale Mb leads to a consideration of the sites through which the rabbit Mb may interact with these antisera. Regions 56--62 and 94--99 are identical in both myoglobins {Fig. 1), and unless their conformations have been altered through amino acid replacements not located in these regions, these two sites would be expected to react completely. With respect to regions 15--22 and 113--119, our previous studies have shown that the binding of reactive regions to antibodies is primarily effected through polar interactions, with non-polar amino acids providing more of a stabilizing role through hydrophobic interactions [ 1 3 ] . Based on these considerations and on the conservative character of the amino acid substitutions in region 15--22 (Ala Is -~ Gly, Va121 -~ Leu) and 113--119 (Arg ~8 -* Lys), these two regions in rabbit Mb would n o t be expected to be completely unreactive. However, it cannot be excluded that subtle conformational changes in these regions directed by these replacements as well as by replacements elsewhere in the rabbit Mb molecule, could diminish the affinity of these sites for their respective antibodies. Although Lys-145 is included in the reactive region 145--151, it plays only a marginal role with some antisera and, of those studied here, is required only for the reaction of the free peptide 145--151 with antiserum G4, b u t is not part of the reactive region in intact, native spermwhale Mb [4]. Therefore, the replacement of Lys-145 by glutamine in rabbit Mb should be w i t h o u t consequence to the reactivity of this region in native rabbit Mb. However, Tyr-151 has been shown to be absolutely essential for the reactivity of this region, and its replacement by phenylalanine completely obliterates its reactivity [ 1 4 ] . Rabbit Mb is, therefore, not expected to react with antibodies to sperm-whale Mb through region 145--151. The expected diminished reactivities for region 15--22 and 113--119 together with the complete unreactivity of region 145--151 would account for the lack of total cross-reactivity of these t w o myoglobins observed by the precipitin inhibition and immunoadsorption results presented above. The foregoing studies clearly indicate that rabbits respond to immunization with sperm-whale Mb by producing antibodies that are directed against regions of the sperm-whale Mb molecule which are both identical {regions 5 6 - 6 2 and 94--99) and closely similar (regions 15--22 and 113-119) to the corresponding sequences in the rabbit's own Mb. Furthermore, these antibodies will extensively crossreact with rabbit Mb through these equivalent regions. As mentioned above both rabbits and goats make antibodies directed against regions of the sperm-whale Mb molecule which are identical in their structures and locations [4--8]. Pertinent, in this regard, is that hen egg-white lysozyme, the second protein whose antigenic structure has been completely determined [ 15--17 ], is also recognized through identical antigenic sites by both rabbit and goat antisera. In a pilot experiment we have also observed that goat Mb is extremely effective in
282 inhibiting the precipitin reaction of sperm-whale Mb with goat antisera to sperm-whale Mb (unpublished results). Although the sequence of goat Mb is not available for comparison at the present time, we predict that similar identities exist between the reactive regions of sperm-whale Mb and the corresponding regions of goat Mb. It is apparent, therefore, that the antigenic sites of sperm-whale Mb are characteristic for sperm-whale Mb, at least with respect to rabbits and goats, and are independent of any sequence identities which m a y occur between sperm-whale Mb and rabbit Mb or sperm-whale Mb and goat Mb. Accordingly, approaches which attempt to localize antigenic sites of proteins by comparing the primary structure of the antigen with that of the homologous protein in the immunized animal [3] will most likely have to be re-evaluated. It is worthwhile to note here that while Mb is an intramuscular protein thought to be sheltered from exposure to the immune system, its presence in normal serum has been demonstrated [18]. The induction of autoreactive antibodies to "sequestered" antigens by immunization with a cross-reacting antigen is not unprecedented (for review see ref. 19). However, the lack of knowledge concerning the molecular structures and locations of protein antigenic sites has lead to uncertainty regarding the precise regions of the proteins through which these cross-reactions are effected. This is the first system to be described in which the structures of the antigenic sites of the cross-reacting protein are completely known, and provides an excellent model for studying some of the molecular aspects of immunologic tolerance to auto-antigens and its termination. The technical assistance of Mr. Duane Richardson is gratefully acknowledged The work was supported by a grant (AM 18920) from the Institute of Arthritis and Metabolic Diseases, National Institutes of Health, U.S. Public Health Service. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
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