Conformational changes induced by hapten in murine monoclonal antibodies to dinitrophenyl groups - the analysis by temperature-perturbation spectroscopy

Immunoh)gl" Letters, 8 (1984) 325 328 Elsevier

lmlet 526

CONFORMATIONAL CHANGES MONOCLONAL ANTIBODIES THE ANALYSIS

INDUCED BY HAPTEN IN MURINE TO DINITROPHENYL GROUPS -

BY TEMPERATURE-PERTURBATION

SPECTROSCOPY

Olga I. LOSEVA I, Vladimir P. ZAV'YALOVL Helmut FIEBIG 2 and Herwart AMBROSIUS 2 IA II- Union Research Institute of Applied Microbiolog.~'. 142200 Serpukhov. Mos~'ow Region. U.S.S.R. 2Sektion Biowissenschqften. KarI- Marx-Universitiit, Leip:ig. G. D. R.

(Received 4 June 1984) (Modified version received 15 August 1984) (Accepted I I September 1984)

1. Summary

Two samples of murine monoclonal antibodies to dinitrophenyl groups were studied by difference thermal perturbation spectroscopy with particular attention to changes in the amount of perturbed chromophores induced in antibodies as a result of hapten binding (~-2,4-dinitrophenyl-L-lysine). Despite the fact that both antibody samples belong to immunoglobulin G1 and have the same type of light chain, K, they were found to differ significantly in the number of the chromophores perturbed by temperature. The binding of hapten decreases the perturbation of chromophores only in the sample with the less rigid structure, as regards thermal perturbation. These data provide evidence that differences in the rigidity of the structure of variable domains affect the extent of conformational changes induced in the antibodies due to the interaction with an antigen. 2. Introduction

Significant differences in the thermal perturbation of tyrosine residues were found previously [1,2] in the Fab fragments of human monoclonal myeloma immunoglobulin G (belonging to the same subclass and having the same type of light chain [1]). This phenomenon was explained by the differences in the K~9" words: hapten trophenyl groups

murine monoclonal antibodies

dini-

0165 2478 / 84 / $ 3.00 © Elsevier Science Publishers B.V.

rigidity of the structure of variable parts of immunoglobulins. In connection with the supposed allosteric mechanism of activation of effector functions of antibodies [3], great interest is drawn to the study of the interrelationship between the rigidity of structure of immunoglobulin domains and the extent of conformational changes induced by the interaction with an antigen. To achieve this purpose, we studied the samples of murine monoclonal antibodies to dinitrophenyl groups belonging to the same lgG1 subclass and having the same type of light chain.

3. Materials and Methods

3.1. Monoclonal antibodies Monoclonal murine antibodies produced by two different hybridomas (BE:< H 2 / F I 2 and BL× H2/El2/E5 [4]) were studied in the present work. 3.2. Purification of antibodies Antibodies were isolated from the ascitic fluid of BALB/c mice by affinity chromatography [5]. 3.3. T.vping of antibodies Antibodies were typed by the antisera to murine IgG subclasses and also to the light chains of K and ,k types. Both of the samples belong to the lgG1 subclass and have the same type of light chain (K). 325

3.4. Homogeneity The homogeneity of antibodies was checked by electrophoresis in 10% polyacrylamide gel in the presence of 0.1% sodium dodecyl sulfate and also by isoelectric focusing in the pH gradient, obtained with the help of carrier ampholytes. 3.5. Difference temperature perturbation spectros-

and 288 nm, induced by the perturbation of tryptophan and tyrosine residues, respectively. The number of the residues perturbed by temperature was estimated using the temperature increment of absorbance [6]: AA~

1 XX=

- - ,

,4 max

At 0

copy Difference spectra were recorded on a spectrophotometer (Hitachi 200/20) in thermostated cells with an optical length of 1 cm. The protein samples under study (0.7 1.0 mg/ml) were dissolved in 0.15 M NaC1 buffered by 0.005 M phosphate at pH 7.4 and poured into two cells: the control cell was kept at 13 °C; the temperature of the sample cell was gradually increased to 45 °C. The difference spectrum in the range 230 520 nm was recorded at every temperature step (the temperature interval between two steps was 3 4°C). 3.6. Calculations The difference temperature-perturbation spectrum of murine lgGl (Fig. 1) displays 2 maxima at 294 AA

0.01-

~rlm 3()O

-O.O1.

where A max is the absorbance of protein solution at the maximum of the absorption spectrum. AA~ is the value of difference spectrum corrected for volume changes occurring upon increasing the temperature, namely

d o d~ AA~ • A A ~ + A 0

a0 where AA~, is the recorded difference spectrum, A o is the absorbance of protein solution at the wave length corresponding to the maximum of tempera-

dodt ture perturbation spectrum,

metric coefficient of thermal increase of the solvent. d 0, d t is the density of solvent at the temperatures of the control and sample cells, respectively. Consequently, the temperature increment of absorbance X~, is the ratio of the absorbance increase at the temperature change by 1° (AAx/At °) to the value of absorbance at the maximum of absorption spectrum Amax. The dependence X~ is the constant value for the given chromophore under the appropriate medium conditions. If ,kJ and ~,~1 are the wave lengths of the maxima of the temperature perturbation spectra of tyrosine and tryptophan residues, respectively, then the number of chromophore residues perturbed by temperature is estimated according to the equation:

6~ax Fig. I. The difference thermal perturbation spectra of murine anti-DNP lgGl (K) antibodies. The solid curve indicates the spectrum of the antibody produced by hybridoma I. The dashed curve denotes the spectrum of the antibody produced by hybridoma II. The samples were dissolved in 5 mM phosphate buffer, pH 7.4 + 150 mM NaCI. The protein concentration was 0.8 mg/m[. The temperature of the control cuvette was 13°C, that of the sample cuvene 37°C.

326

is the volu-

do

xxPr" "A u)(Trp "A lIXpr .

xTrp

~Tyr max

xTyr ~,Trp "'M " "Au

pr ~max

Xx" "'M

xpr. ~,Tyr "'M "'X"

~Trp max

~,Tyr. yTrp "'M "A u

3¢Tyr. 3(Trp "M I "M

pr . yTyr

x T y r . xTrp "An "'hi

(-~ax' 6Tyr max' ~lrp max are the molar extinctions of the solutions of protein, tyrosine and tryptophan, respectively, at the maximum of the absorption spectrum, a,/3 are the number of tyrosine and tryptophan residues perturbed by temperature. Xpr, yXyr ~,Vrpare the in"'X '"X crements of absorbance of the protein and chromophores (tyrosine and tryptophan) in model solutions at the wave length X. The temperature perturbation spectrum of murine monoclonal antibodies was modelled by tyrosine solution in 30% ethylene glycol (maximum at 288 nm) and by tryptophan solution in 50% ethylene glycol (maximum at 294 nm). The total number of tyrosine and tryptophan residues in the molecules of murine lgGl was determined spectrophotometrically according to Edelhoch [7]. Fifty-five tyrosine residues and 20 tryptophan residues were found.

4. Results

The data of difference thermal perturbation spectroscopy of anti-dinitrophenyl antibodies are presented in Figs. ! and 2 and Table 1. According to these data the antibodies produced by two different hybridomas possess different properties. The antibodies produced by hybridoma 1 have a more labile structure (Fig. 1): 88% of tyrosine and 64% of tryptophan residues are perturbed by temperature; also, conformational properties of these antibodies are changed as a result of binding of the hapten, ~-2,4-DNP-lysine (Fig. 2). The thermal perturbation of tyrosine residues is decreased by 16% or by 9 residues. The thermal perturbation of tryptophan residues changes less significantly. The antibodies produced by hybridoma II are less sensitive to thermal perturbation (Fig. 1): 64% of tyrosine residues and 41% of tryptophan residues are perturbed. Moreover, the hapten binding does not affect the thermal perturbation of chromophores in the antibody sample (see Table 1).

5. Discussion

According to the data of difference thermal perturbation spectroscopy, tyrosine residues perturbed by temperature in the samples of murine monoclo-

AA

0.01-

<

~. 11111

sbo " . . . . . . . . . . abo

sb(

-O.Ot"

Fig. 2. The difference thermal perturbation spectra of monoclonal anti-DNP IgGI antibody (hybridoma I). The solid curve denotes the spectrum of the free antibody. The dashed curve indicates the spectrum of the antibody mixed with hapten (~-2,4-DNP-L-lysine) at molar ratio 1:2.5 (about 90% saturation of the active sites). The samples were dissolved in 5 mM phosphate buffer, pH 7.4+ 150 mM NaCI. The protein concentration was 0.8 mg/ml. The temperature of the control cuvette was 13 °C, that of the sample cuvette 37°C.

nal antibodies under study are located in a microenvironment, the polarity of which corresponds to 25-40% ethylene glycol, whereas 45- 50% ethylene glycol corresponds to the environment of tryptophan Table 1 The data of difference temperature perturbation spectroscopy of monoclonal murine antibodies to dinitrophenyl groups Protein

BLxH2/FI2 (hybridoma 1) + DNP-lysine MR 1:2 BLxH2/EI2/E5 (hybridoma 11) + DNP-lysine MR 1:2

Temperature perturbation of tyrosine residues

Temperature perturbation of tryptophan residues

%

Q-ty

%

Q-ty

88_+2.0

49+1.0

64+2

13_+1

72_+2.0

40_+1.0

58_+2

12_+1

64_+2

36+1

40_+2

8_+1

68+2

37_+1

41+2

8+1

327

residues. This correlates with the data of X-ray analysis [8, 9] which indicate that a significant part of tyrosine and tryptophan residues in immunoglobulins are located in the cracks in the region of contacts between domains, bulky hydrophobic side chains of tryptophan residues often being found in the hydrophobic cores of domains. The compared samples of monoclonal antibodies significantly differ in the number of tyrosine and tryptophan residues perturbed by temperature. In connection with the fact that both samples belong to the same IgGl subclass and have the same type of the light chain K, these data indicate the differences in the rigidity of structure of variable parts of antibodies. Similar phenomenon was observed previously for monoclonal human myeloma immunoglobulins G [1, 21. The hapten binding decreased the thermal perturbation of tyrosine residues only in one of the samples of monoclonal antibodies characterized by the less rigid structure. The number of conservative tyrosine and tryptophan residues in the variable regions of murine IgGl was found to be (2×9) = 18 and (2×5) = 10, respectively [10]. This amount can account for the change observed in the case of sample I of monoclonal antibodies. The binding site of antiDNP antibodies usually contains a great number of tyrosine and tryptophan residues, e.g. in the case of MOPC 315, Tyr 33, 104 of the heavy chain and Tyr 34, Trp 93 of the light chain are involved in the binding of the DNP hapten and are protected by direct shielding effect from solvent perturbation [11]. With regard to thermal perturbation in MOPC 315, the rigidity of the microenvironment of not more than 1 Tyr and i Trp residue increased as a result of hapten binding [12]. No changes in thermal perturbation of Tyr and Trp residues were observed in the case of the sample I1 of murine anti-

328

DNP monoclonal antibodies (see Table 1) and non-precipitating pig anti-DNP antibodies [13]. In all the cases no changes of the positions of maxima of difference spectra were observed. Thus, the decrease of the extent of tyrosine and tryptophan maxima in thermal perturbation spectra indicates the increase of the rigidity of the microenvironment of the chromophores (conformational effect) rather than the direct shielding effect against solvent perturbation. On the basis of these data one may conclude that the extent of conformational changes induced in the antibodies due to the hapten binding depends on the rigidity of structure of the variable domains.

References [1] Zav'yalov, V. P. (1978) Ph.D. Thesis, l,enin State Library, Moscow, pp. 48 151. [2] Abramov, V. M., Arkhangelskaya, Z. A. and Zav'yalov. V. P. (1983) Biochim. Biopbys. Acta 742, 295 302. [3] Metzger, H. (1974) Adv. lmmunol. 18, 169. [4] Fiebig, H. (1981) Doctoral Thesis, Karl Marx University, Leipzig, pp. 61 69. [5] Fiebig, H. and Ambrosius, H. (1975) Acta Biol. Med. (German) 34, 1681 1695, [6] Demchenko, A. P. and Zyma, V. L. (1975) Studia Biophys. 52, 209 221. [7] Edelhoch, H. (1967) Biochemistry 6, 1948 1954. [8] Poljak, R. I., Amzel, L. M. and Chert, B. L. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 3440 3444. [9] Deisenbofer, J. (1981) Biochemistry 20, 2361 2370. [10] Feinstein, A. and Beale, D. (1981) in: Structure and Function of Antibodies (L. E. Glynn and M. W. Stewart, Eds.) pp. 148 199, J. Wiley and Sons, Chichester. [I 1] Dwek, R. A. (1977) Contemp. Top. Mol. lmmunol. 6, I 52. [12] Zav'yalov, V. P., Abramov, V. M., Skvortsov, V. G. and Troitsky, G. V. (1977) Biochim. Biophys. Acta 493, 359 366. [13] Loseva, O. 1., Abramov, V. M., Zav'yalov, V. P.. Troitsky, G. V., Olgovska, Z. and Fran~k. F. (1982) Eur. J. Biochem. 121,631 635,