Quantitative immunoligical studies on single amino acid substitution in human hemoglobin: Demonstration of specific antibodies to

lmmunoehem~stry,1974,Vol 11,pp. 21-27 PergamonPress Printedin GreatBritain

QUANTITATIVE I M M U N O L O G I C A L STUDIES ON SINGLE A M I N O ACID SUBSTITUTION IN H U M A N H E M O G L O B I N : D E M O N S T R A T I O N OF SPECIFIC ANTIBODIES TO MULTIPLE SITES* MORRIS REICHLINt Departments of Medicine and Biochemistry, Veterans' Administration Hospital, SUNY at Buffalo School of Medicine, Buffalo, New York, U.S.A. (Received 13 June 1973)

Abstract--Rabbit antibodies have been elicited to many mutant human hemoglobins. These antimutant sera distinguish A 1 hemoglobin from the mutant hemoglobins more regularly and by larger quantitative differences than these immunological distinctions are made by rabbit anti AI sera. Of 14 variant hemoglobins injected into rabbits, 12 regularly elicited mutant specific antibodies. These data are discussed in terms of a hypothesis relating binding energy differences between A 1 and mutant hbs reacting with specific antibody and the amino acid sequence differences known to exist between human and rabbit hemoglobins.

INTRODUCTION

MATERIALS

In a previous paper (Reichlin, 1972) one amino acid mutants and other defined variants of human hemoglobin (hb) were compared to normal hbA 1 with rabbit antiA1 sera in quantitative complement fixation experiments.:~ It was found that a number (7) of the mutants could be distinguished from A1 with antiA: sera while an even larger number (11) were undistinguishable from A v Moreover, it was found that distinguishable mutants of A~ were located in regions of the primary sequence where human and rabbit hbs have many sequence differences and the undistinguishable mutants were restricted to regions where human and rabbits hbs possess identical sequences. This report describes the results obtained when several one amino acid mutant hbs are injected into rabbits and the resultant antisera are assayed for antibodies which can distinguish the mutants from A 1. One frequently obtains antisera specific for the difference between the mutant and A1 ; in addition, these antimutant sera distinguish mutants from A t more easily and consistently than antiA1 sera distinguish A~ from mutants.

AND METHODS

Collection of mutant hbs, their purification, characterization, and sources have been previously described (Reichlin, 1972). Hbs New York ~13....... and Arora, fl22....... were obtained from Dr. Helen Ranney of the department of Medicine, SUNY at Buffalo, Buffalo, New York. Quantitative complement fixation, quantitative precipitin analysis, and a discussion of the significance of the errors were described in a prior publication (Reichlin, 1972). Preparation of rabbit antisera to human hbs was described previously (Reichlin et al., 1964). Goats were immunized with human hbA 1 by a schedule, which had been developed for polymeric human cytochrome c (Reichlin et al., 1970). RESULTS

Immunization with mutants from the variable regions of hb Comparison of the primary sequences of rabbit and human hbs illustrates the nonrandom character of the variation in the sequences between the two species (Reichlin, 1972). According to the designation previously adopted, that half of the sequence containing 90 per cent of the sequence variation of the two species is called 'variable' and the remaining half which contains only 10 per cent of the sequence variation between the two species is called 'conservative'. Rabbits were immunized with mutants from both variable and conservative regions of the hb sequence. In Table 1 are listed the results of two kinds of quantitative measurements of the antigen-antibody reaction utilizing antisera arising from immunization of

* Work carried out during tenure of Career Development Award 5KO3-AM20729. t Supported by a Grant from the United States Public Health Service AM10428 and designated funds from the Veteran's Administration. :~For purposes of brevity and to avoid excessive redundancy 'A 1' will be used to denote A~hb and 'mutant(s)' for mutant(s) hb(s). 21

22

M. REICHLIN Table 1. Comparison of A 1 and mutants with anti mutant sera: mutants from variable regions of Hb sequence Precipltin #g Abn/ml

Serum

C Flxatmn Ratio Cns0 units fixed

AI 65 140 56 367 50 141

S 84 180 72 434 70 158

A1/S × 100 79 78 78 85 71 89

A1S × 100 70 74 92 88 50 86

A2 53 104 85 C 47 19 67 98 Korle Bu 50 56 54 57

A1/A 2 x 100

A1/A 2 × 100

270 271 272 273

A1 30 41 63 A1 16 13 48 63 A1 44 54 34 47

57 40 74 A1/C x 100 34 68 72 64 A1/KB x 100 88 96 63 83

61 63 62 AI/C × 100 52 61 67 69 A1/KB x 100 78 94 78 50

AI

Joxford

240 241 242

118 90 232

152 123 286

Ax/Jo x 100 78 73 81

Al/Jo x 100 70 75 80

A1 114 A1 41 41 47 A~ 11 25 A1 68 31 275

Iaurl,ngto.

A1/1 x 100

Aa/1 x 100

135 Cm~l~ 51 55 63 Arora 21 39 NY 85 42 300

84 AI/Cu x 100 80 75 75 A1/Arora × 100 52 64 AI/NY x 100 85 74 91

211 212 261 262 301 323 78 79 80 229 230 231 429

216 207 208 209 417 418 337 338 339

rabbits with m u t a n t s from the variable regions. M u t a n t and Aa hbs were compared by b o t h quantitative precipitin analysis a n d quantitative complement (C) fixation with rabbit antisera resulting from immunization with the respective m u t a n t hb. The maximally precipitable a n t i b o d y (Ab) nitrogen (N) per ml of serum for b o t h m u t a n t and A~ hbs a n d the ratio of these values for each a n t i m u t a n t h b serum are listed. In the case of the C fixation experiments, only the ratio (A1/mutant) of the m a x i m u m C fixed is listed. In every case the serum was diluted such that the m u t a n t h b fixed from 50-70 per cent of the available C for reasons previously discussed (Margoliash et al., 1970). Every antiserum distinguishes the m u t a n t from A~ by both the precipitin and C fixation techniques. O n the average, b o t h techniques measure the specificity of the m u t a n t as compared to A 1 by a b o u t the same

89

A/Ca x 100 32 41 52 Al/Arora × 100 80 43 A1/NY x 100 99 95 90

extent. The mean value of the A1/mutant ratio is 73.5 per cent for precipitin tests a n d 70"5 per cent for the C fixation technique. R a b b i t antisera made to hbA 2 show more specificity for A2 as compared to A1 than do sera made against the one a m i n o acid mutants. Since there are 10 amino acid sequence differences between A 2 and A 1 (See Table 6) this slightly larger Az-A 1 immunological difference is not surprising. In the case of the double m u t a n t Cmr~em there are very large differences between A1 a n d Cnarl~r, noted particularly in C fixation tests with the antiCmr~em sera. It is likely that this reflects specific antibodies directed against both mutations existing in hbCmrle m. As seen in Table 6, Cnar~emcarries valine instead of glutamic acid at t 6 and asparagine for aspartic acid at fl73. Thus Cmrlcm is like hbS at t 6 a n d like hb Korle Bu at fl73.

23

Antibodies to Mutant Human Hemoglobins

reciprocal of the result previously reported with antiA t sera (Reichlin, 1972) in which it was found that hbS was distinguishable from hbA1 by 15 per cent and Cmr~em was distinguishable from At by 30 per cent in C fixation.

50-

40--

==

:t

Immunization with mutants from the conservative regions of hb

50-

20--

I0--

I 1.6

0 0

I

I

I

3.2

4.8

6.4

p.g Ag N odded/ml serum

Fig. l. Quantitiative precipitin test of rabbit serum 209B2, antiCm,~,m with mcrements of hemoglobins Cm,],,, (O), S (O), and At(O ) added.

Figure 1 demonstrates these effects as Cmr~em, S, and At are compared in their ability to precipitate antibody from one antiCmrlem serum. As can be seen, hbS is distinguishable from hbCna~l~m and hbA 1 is even more antigenically deficient to Cmr~ m than hbS. This is the

In Table 2 are tabulated the data from 14 rabbits immunized with 5 one amino acid mutants located in portions of the hb sequence where rabit and At hbs have identical sequences. In contrast to the results obtained with sera obtained by immunization with mutant hbs from the variable region (where mutant specific antibodies are regularly observed) mutant specific antibodies were elicited with some mutant hbs from the conservative regions and not with others. For example, animals immunized with hbs MMi]waukee and LF~..... (Reichlin et al., 1970) produced antisera which failed to show significant differences between the mutants and At in precipitin tests and only occasionally in C fixation tests. The serum from one animal in each group (341 and 265) showed a small C fixation difference when the mutant and A t were compared. With the antisera elicited by hb Manitoba, all three sera showed a significant precipitin difference but sera 297 and 298 could not distinguish A t from Manitoba in the C fixation technique. Similarly, with antisera raised against hb Riverdale, serum 226 cannot distinguish Riverdale from At by either test, serum 227 is more reactive with Riverdale than A t by precipitation but not by C fixation, while only serum 228 can distinguish Riverdale from At by either test. Thus, mutant hbs from the 'conservative' regions often do not elicit

Table 2. Comparison of mutants and A 1 with anti mutant sera: mutants ~om conservative regions of Hb sequence Precipitin analysis #gAbN/ml

Ratio CHs0 Umts Fixed

340 341 342

A1 364 86 117

MM,I 368 89 118

AI/MM,I x 100 99 97 99

A/MMIL x 100 97 90 100

A1 144 87 60

Lv~ 152 89 59

A1/LF~ x 100 95 98 101

A1/Lv~ x 100

264 265 266

A1 15 56 45 A1 226 63 19 A1 37 121

Man 23 69 53 RIV 213 94 29 CBZ B 70 117

A1/Man x 100 65 81 85 A/R,v X 100 106 68 65 A1/CBZ B x 100 53 103

A1/Man x 100 96 100 78 A/RIv X 100 95 100 43 A1/CBZ B x 100 78 89

297 298 299 226 227 228 255 256

95 91 96

M. REICHLIN

24

Table 3. Tabulated precipitin data with several rabbit antiHbA1 sera: comparative antigenicity of one amino acid mutants and variants of human Hb with HbA~ Serum No. Human Hb A1 S C Korle Bu Cmrlcm I

Joxford A2

81

85

87

89

85 85 83 74 74 83 83 79

58 59 57 58 57 50 52 54

184 185 189 ---

220 215 218 -20__.88 218 214 16.__33

--

---

Table 4. Tabulated precipitin data witl~ two goat antihuman HbA~ sera. Comparative antigenicity of one amino acid mutants and variants of human Hb with HbA~ Serum No.

All values are in/~g Ab N per milliliter serum.

mutant specific antibodies and in other cases produce antibodies which have the interesting property that they can distinguish the mutant from At in precipitin tests but not in C fixation reactions (and vice versa).

Precipitin tests of human hbs with rabbit and goat antihbA t Sera With a single rabbit antihbA t serum, it had previously been reported that by quantitative C fixation techniques, hbS was shown to be antigenically deficient to hbAt (Reichlin et al., 1964). It was also pointed out that quantitative precipitin analysis with the same rabbit serum failed to show an antigenic difference between At and S. Subsequently, a large number of mutants were compared to At with rabbit antiAt sera in C fixation tests (Reichlin, 1972). It was of interest to know if these antiA1 sera would be less able to distinguish mutants from A~ by quantitative precipitin than by C fixation analysis. Table 3 lists the maximally precipitable Ab N per ml of the mutants which can be distinguished from A t by such sera in C fixation analysis. It is seen that such mutants are in general not distinguishable from A~ by precipitin tests. Hbs S and C

Human Hb A1 S C Korle Bu

CHarlem I Joxford A2 Riverdale New York CBZ B Arora LF......

4

5

686 65___~3 600 688 62____44 698 694 48.___33 679 675 716 64..~4 696

528 511 528 518 49__22 525 511 49___~1 520 550 515 515 523

All values are in #g Ab N per milliliter serum. are seen as identical to At with all four rabbit sera and I and Joxfora are identical to At with two of the three sera used. Moreover, when the rabbit serum does significantly distinguish the mutant from At by the precipitin tests (those values that are underlined), the mean ratio of mutant/At precipitin values is only 0.88 compared to 0.735 for the A1/mutant ratio for precipitin tests with rabbit antimutant hb sera. Table 4 shows the results obtained by comparing the antigenicity of hbA1 and 12 of the variant hbs with antisera produced against At in goats. As with the rabbit antiAt sera few of the variants can be distinguished from A~ with goat antiA~ in the precipitin test. With goat 5 only hbs CmrLem and A2 are distinguishable from A t and the differences are small, being 7 per cent in both cases. With goat 4, A 2 is easily distinguishable from At precipitating only 70 per cent of the total antibody. The differences exhibited by hbs S, C, Arora, and CHarlem in precipitin behavior from A t are small and they precipitate 95, 88, 94 and 91 per cent respectively

Table 5. Quantitative precipitin AntiA 1 sera Mutants from variable regions Mutants from conservative regions Antimutant sera Mutants from variable regions Mutants from conservative regions (a) M Milwaukee (3) L Ferrara (3), and one Riverdale animal (b) 2 Manitoba animals and one Riverdale animal

Quantitative C 'fixation

__+u

+a

+

+

+

"All data concerning C fixation with rabbit antiAx sera have been published in J. molec. Biol. 64, 485 (1972). b Most mutants are not distinguishable from A~ by precipitin tests and those that are distinguishable show significant but small differences from Ax.

25

Antibodies to Mutant Human Hemoglobins Table 6. Summary of substitutions in hemoglobin variants fl Variants

S C Arora Riverdale

MMilwaukee

Korle Bu New York

CHarlem A2

Sequence position

A~ to variant change

6 6 22 24 67 73

glu to val glu to lys glu to gln gly to arg val to glu asp to asn val to glu. glu to val asp to ash asn to thr thr to asn glu to ala thr to set ala to set thr to gin his to arg his to asn val to gin gin to met

113

6 73 9 12 22 50 86 87 116 117 125 126

of the total antibody. Thus the goat antiA t sera while differing somewhat from the rabbit antiA1 sera in detailed specificity behave similarly in their capacity to discriminate between various mutant and At hbs. Finally, Table 5 summarizes and contrasts the ability of antiA1 sera to distinguish mutant hbs from At with the ability of antimutant sera to distinguish A1 from the mutants. It is seen that with antiA1 sera, mutants from the variable regions are regularly distinguished from A~ only by the C fixation technique. Quantitative precipitin analysis only sporadically distinguishes mutant hbs from At and then the differences are small. Mutants from the conservative regions of the hb sequence are not distinguishable from A~ with antiA~ sera by either C fixation or precipitin tests. The antimutant sera resulting from immunization with mutants from the variable region in every case contain large amounts of antibodies more specific for the mutant than for At demonstrable by either the precipitin or C fixation technique. When mutants from the conservative regions of hb are injected into rabbits, several different results are obtained. With some m u t a n t s (MM,lwauke e and Lv...... ) little or no mutant specific antibody is found. With other mutants (Manitoba, Riverdale) there is great variation in response even in a small group, but here one sees in several rabbits antibodies which distinguish Ax from the mutant Hb more easily by the precipitin test than by the C fixation technique. This capacity of a serum to distinguish an antigenic difference by precipitin tests and not by C fixation tests has been seen primarily thus far with sera arising from immunization of rabbits with mutants from the conservative regions of hb.

~t Variants

Joxford Ia.rhngto n LF ......

Manitoba CBZ B

Sequence position

A1 to variant change

15 16 47 102 141

gly to asp lys to glu asp to gly ser to arg arg to o

DISCUSSION The observations comprising this report raise three questions which can be stated as follows: (1) What are the reasons that mutants from the variable regions give rise to antisera which more easily distinguish At from mutants than antiAt sera distinguish mutants from At ; (2) What accounts for the fact that some mutants from the conservative regions of hb give rise to mutant specific antibodies and others do not; and finally (3) What are the reasons underlying the different abilities of various quantitative immunochemical methods to detect the specificity related to single amino acid changes in various sera resulting from immunization with either At or mutant hbs. While definitive answers to these questions await experimental facts, it is perhaps useful to discuss these questions by correlating the known facts of amino acid sequence relationships with the immunochemical reactions. The first question can be viewed from the following perspective. When one immunizes an animal with At, antibodies arise which are complementary to an aspect of primary structure in the hb sequence which can be correlated with the differences in sequences between rabbit and human hbs (Reichlin, 1972). These so called 'variable' regionsin alllikelihoodcontain the amino acid residues which constitute the antigenic determinants. The mutants distinguishable from A1 are all found in such 'variable regions' and are selected for assay only because of their availability. Remarkably, all mutants examined thus far can be quantitatively distinguished from A1 in C fixation reactions with rabbit antiAt sera. It is quite conceivable that the major part of the binding of antigen in the determinant for example related

26

M. REICHLIN

to HbS (f16 ........ ) is not related to position 6 but perhaps to fl positions 4, 5, 7 or 8 (or some combination of these residues). Substitutions at any of these sequence positions might have a more profound effect on the difference in binding of such a modified Hb compared to A~ than the difference actually measured between hbs A and S with antiAa. In the reciprocal situation, when one immunizes a rabbit with hbS, one operationally selects for antibody which is more dependent on position f16 than when hbA1 is injected (where the immunodominantresidue may be one other than j~6) giving rise to the greater difference in antigenic reactivity between Hbs S and A~ with rabbit antiS than the comparable reactivity between hbs Ax and S with rabbit antiA1. This greater specificity for hbS would be predictable from the tenets of the clonal selection theory (Burnet, 1959), and need not be viewed as a novel or ad hoc premise(s). This greater reactivity might speculatively be related to a larger increment in binding energy between S and Aa reacting with antiS as compared to A 1 and S reacting with antiA v Such hypothetical binding differences can be correlated with the sequence differences between the antigen and the structure of that protein in the immunized animal. That is, the fl N terminal sequence differences between fls and rabbit fl are greater than those between flA and rabbit ft. In the former case there are three differences between fls and rabbit fl positions 4, 5 and 6) while only two such differences exist (positions 4 and 5) between flA and rabbit ft. In all the mutant hbs studied here, there is a greater mutant hb-rabbit hb sequence difference than the corresponding sequence difference between A~ and rabbit hbs. It is tempting to think it is the quality and quantity of such sequence differences between the antigen and the same protein in the immunized animal that determines at least in part the specificity of antibodies elicited to protein antigens which are possessed by the immunized animal and to which the animal is normally tolerant. Such sequence differences would constitute a quantitative expression of what it is to be 'foreign' in the self-nonself discrimination animals make in the preservation of their naturally tolerant state. Studies are in progress to determine the binding energy differences hypothesized to exist between such pairs of hbs differing by a single amino acid in their reactions with specific antibodies. In addition, studies are also underway which relate such binding differences to sequence differences between the antigen and the same protein in the immunized animal. The second question raised concerns the heterogeneity of effects seen when mutant hbs from the conservative regions are injected into rabbits. With two of the mutant hbs studied, the substitution does not involve a surface residue. These are Hbs MMi~*,auke~and Riverdale. Thus, it is not surprising that the rabbit sera raised against the M~i~waukee protein (Reichlin et al., 1970) do not distinguish Hb MMi~waukeefrom HbA~. The surface of the MM,~wa,ke~protein must appear to the immunological recognition apparatus (and the se-

creted recognition product, antibody) to be identical to the surface of hbA 1. Indeed, such data indicate that what the immunological recognition apparatus "senses' is the protein surface and not the linear sequences per se. In the case of hb Riverdale the response is heterogeneous even among the three animals studied. One rabbit serum (226) has no antibody which distinguishes Riverdale hb from Alhb, while two other rabbits do have 'Riverdale' specific antibody which in one case (227) is demonstrable by precipitation but not by C fixation. The surface of hb Riverdale may not be entirely normal as this is a protein thought to be made unstable by the introduction of a bulky gaunidino group for a hydrogen atom at the point of close proximity of the B and E helices (Ranney et al., 1968), Apparently some rabbits see the probable surface distortion related to this internal substitution and others do not. Lastly, Hb L F ...... elicits little antibody that distinguishes this mutant from hb A1. Since the substitution in LF...... is glycine for histidine at position 47, the substitution involves loss of an imidazole side chain. Thus, the loss of such a group seems not to be provocative immunogenically.Whether most internal substitutions and the replacement of bulky side chains by hydrogen atoms will be immunologicallysilent awaits the study of many such cases. Lastly one might ask why with certain sera (antiA 1 for example) complement fixation reactions can detect differences between closely related antigens while the quantitative precipitin reaction sees the same two antigens as identical. One might also wonder why with rabbit sera raised in rabbits against certain mutants from the conservative regions, such sera detect an antigenic difference between the mutant and A1 in precipitin tests but not in C fixation reactions. One can only speculate about such phenomena but preliminary data suggest that the first case is best explained by a small difference in binding affinity between the two antigens where the antibody involved possesses high affinity while in the second case a small difference in binding affinity exists but the antibody is of much lower intrinsic affinity. Because C fixation reactions are carried out at molar concentrations of reactants of 10 -9 to 10-10 M/L and precipitin reactions at 10 ~ to 10 - ° M / L the former technique will detect small differences in pairs of antigens reacting with antibodies with dissociation constants (for such antigens) in the range of 1 0 - 9 o r 10- ao M/L while precipitin reactions will more sensitively detect antigenic differences where the intrinsic dissociation constant is in the range of 10- 5 to 10- 6 M/L. Other possibilities exist to explain such differences in the ability of different immunological tests to ascertain antigenic differences. However, they are quite complex and since the detailed mechanism is subject to straightforward experimental analysis (which is underway) it is perhaps best to leave the argument in its present speculative framework. Finally, it should be stated that whatever the underlying mechanism it has been repeatedly demonstrated in this study that the most sensitive and consistent

Antibodies to Mutant Human Hemoglobins method for the immunological detection of single amino acid changes in a related series of protein antigens is by immunization with each mutant protein. In a high proportion of variant hbs, mutant specific antibodies are obtained. Acknowledgements--The author is appreciative of the expert technical assistance provided by Mrs. Jacquelyn Hill, Mrs. Mary Ahl, and Miss Nancy Bailing. REFERENCES

Burnet F. M. (1959) The Clonal Selection Theory of Acquired Immumty. Vanderbilt University Press, Nashville, Tenn.

27

Margoliash E., Nisonoff A. and Reichlin M. (1970) J. biol. Chem. 245, 931. Ranney H. M., Jacobs A. S., Udem L. and Zalusky R. (1968) Btochem. biophys. Res. Comm. 33, 1004. Relchlin M. (1970) Immunochemistry 7, 15. Reichlin M. (1972) J. molec. Biol. 64, 485. Reichlin M., Hay M. and Levine L. (1964) Immunochemistry 1, 21. Reichlin M., Nisonoff A. and Margoliash E. (1970) J. btol. Chem. 245, 947.