Theoretical and experimental epitope mapping of thymosin β4

ELSEVIER

JOURNALOF IMMUNOLOGICAL METHODS Journal of ImmunologicalMethods 177 (1994) 131-137

Theoretical and experimental epitope mapping of thymosin / 3 4 S. Becker a,., F.P. Armbruster b, B. Miiller b, H. Echner a, A. Kapurnotu a, E. Livaniou a, M. Miheli6 a, S. Stoeva a, W. Voelter a a Abteilung fiir Physikalische Biochemie, Physiologisch-chemisches Institut der Universitiit Tiibingen, Hoppe-Seylerstr. 4, D-72076 Tiibingen, Germany b Immundiagnostik GmbH, Wilhelmstr. 7, 64625 Bensheim, Germany

Received 1 July 1993;revised 18 May 1994;accepted 22 July 1994

Abstract Two rabbit polyclonal antisera, one directed against thymosin /34 and the other one against the peptide fragment thymosin /34 (1-14) were characterised by epitope mapping. Hexapeptides representing the whole sequence of the native peptide and overlapping by one amino acid were synthesised on polystyrene pins. The antigenic determinants were identified in microtitre plates with an ELISA procedure. The polyclonal serum against thymosin /34 detected three epitopes (N-terminal, mid-region and C-terminal) whereas the polyclonal serum against the fragment contained only antibodies specific for the N-terminal epitope. These experimental results are consistent with theoretical predictions. Keywords: Thymosin/34; Epitope mapping; Antigenic determinant; Thymus peptide

1. Introduction The thymus is one of the main sites for the production of different thymus peptides (thymus hormones, thymus factors). Due to their different isoelectric points thymosins are classified as a-, /3- and ~/-thymosins. An important peptide of the /3-thymosin family is thymosin /34, a 43 amino acid residue peptide with a molecular weight of

Abbreviations: BSA, bovine serum albumin; KLH, keyhole limpet hemocyanin; SDS, sodium dodecyl sulfate; HRP, horseradish peroxidase; DMF, dimethylformamide;OPD, ophenylenediamine dihydrochloride; PBS, phosphate-buffered saline. * Corresponding author.

4963 Da, and an isoelectric point of 4.6 (Heinzel and Voelter, 1983; Miheli6 and Voelter, 1994). The primary structure is shown in Fig. 1. The tetrapeptide thymosin f14 (1-4) is most probably produced by proteolysis from thymosin /34. It has an inhibitory effect on the proliferation of hemopoietic pluripotent stem cells (Grillon et al., 1990). Because of the sequence homology to other peptides of the/3-thymosin family (Fig. 2) it is necessary to produce antibodies specific for distinct epitopes of thymosin /34, in order to develop specific immunoassays. The antigenic sites of peptides and proteins can be predicted using the parameters of hydrophilicity, flexibility or amino acid side chain surface probability (Voelter et aI., 1990c). However, no single param-

0022-1759/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0022-1759(94)00216-9

132

S. Becker et al. /Journal of Immunological Methods 177 (1994) 131-137

A¢-N

H

5

~

10

2. Materials and methods

2.1. Reagents

COOH ~

40

Fig. 1. Primary structure of thymosin f14. eter is sufficient, Jameson and Wolf (1988) have developed a computer program to predict potential antigenic determinants directly from the primary amino acid sequences of polypeptides and proteins integrating hydrophilicity (H), surface probability (S), backbone flexibility (F) and secondary structure (CF: Chou-Fasman; GR: Garnier-Robson) parameters based on the following equation: N

A i = • 0 . 3 ( H ) + 0.15(S) + 0.15(F) + 0.2(CF) i=1

+ 0.2(GR) These predictions for thymosin /34 have been compared with the results of two dimensional NMR experiments using the naturally occurring molecule (Zarbock et al., 1990) and epitope mapping. Classical methods for the identification of the antibody binding sites of peptides, for example PTH, have been performed using lzSI-labelled fragments of PTH in binding studies (Zanelli et al., 1983) and additionally studying the exchange of labelled and unlabelled fragments (Fischer et al., 1974; Visser et al., 1979; Hehrmann et al., 1980; Atkinson et al., 1982). In another approach to epitope mapping recombinant DNA is used, exchanging or deleting amino acids at the genetic level (Van Duijnhoven et al., 1991). TI34: AcSDKPDMAEIE-KFDKSKLKKT-ETQEKNPLPS-KETIEQEKQA-GES T139: AcADKPDLG~EIN-SFDKAKLKKT-ETQEKNTLPT-KETIEQEKQA-K T[310: AcADKPDMGEIA-SFDKAKLKKT-ETQEg~TLPT-KETIEQEKRS-EIS Fig. 2. Amino acid sequences of selected /3-thymosins.

N,N-dimethylformamide (DMF), piperidine, trifluoroacetic acid, triethylamine, phenol, diisipropylethylamine, BSA and OVA were supplied by Serva (Heidelberg, Germany). Dichloromethane, Tween 20, sodium dodecylsulfate (SDS) were purchased from Sigma (Munich, Germany) and ethanedithiol from Aldrich (Steinheim, Germany).

2.2. Buffers The ELISA buffer used was PBS (0.14 M NaCI-2.7 mM KC1-1.5 mM KH2PO4-7.1 mM NazHPO4, pH 7.4), containing 1% (w/v) ovalbumin, 1% (w/v) BSA and 0.1% Tween 20. PBS/0.05 % Tween 20 was used as washing buffer. The substrate solution (pH 5.0) contained 0.1 M citrate buffer/0.03% H 2 0 2 / 600 mg O P D / I (Dako, Hamburg, Germany).

2.3. Antisera The two polyclonal antisera directed against /34 and thymosin /34 (1-14) were obtained from the fresh blood of two rabbits. Synthetic thymosin/34 and thymosin /34 (1-14), both coupled to KLH with glutardialdehyde were used as antigens. These peptides were synthesised using the Fmoc/Bu t strategy and purified by HPLC (Voelter et al., 1990a, b). Blood samples were taken after the basic immunisation and multiple booster injections. Epitope mapping was performed only with antisera showing a titre over 1/3000 in a competitive solid phase ELISA (Miheli6 et al., 1992).

thymosin

2.4. Epitope mapping Epitope mapping was performed using a kit from CBS, Cambridge, UK. Hexapeptides representing the whole sequence of thymosin /34 were synthesised on a polystyrene pin block. Peptides overlapping by one amino acid were produced on pins with the following sequences: pin 1, amino

S. Bec ker et al. /Journal o f Immunological Methods 177 (1994) 131-137

acid 1-6; pin 2, amino acid 2-7; etc. The synthesis were performed according to the method of Geysen et al. (1984), using Fmoc-activated amino acids. These reagents were of high grade purity, designed for solid phase peptide synthesis using DMF as solvent. After completion of the synthesis, the N-terminus of each peptide was acetylated, to remove the unnatural charge from the N-terminus. For this step the following acetylation mixture was used: DMF:acetic anhydride:triethylamine, 5:2:1 (v/v/v). All protecting groups of the side chains were removed from the synthetic products by treating the pins with the following cleavage mixture: trifluoroacetic acid:phenol:ethanedithiol, 95:2.5:2.5 (v/v/v).

2.5. Determination of antibody binding in an ELISA The ELISA procedure was carried out in duplicates in the wells of microtitre plates (Nunc, PLOTSTRUCTURE of:

T'ny~e|n- Beta4

t h y m - b e t a 4 . p e p ck:

,z

tJB6

133

Roskilde, Denmark). Prior to the test the pin block was cleaned in an ultrasonic bath with SDS buffer for 0.5 h, then rinsed with hot water and methanol. All further assay steps were carried out in the wells. First the pins were incubated with 250 ml ELISA buffer for 1 h at room temperature, to block free binding sites and to prevent unspecific binding. Then the pins were incubated with 175 ~1 antiserum (diluted 1/500 in ELISA buffer) at 4°C for about 20 h, washed for 3 × 5 min with 250 ~1 washing buffer and incubated for 1 h at 25°C with 175 ~1 of the second antibody solution (swine anti-rabbit-HRP; Dako, Hamburg; diluted 1/500 in ELISA buffer), washed again and incubated with 250/xl of substrate solution until sufficient coloring was observed. The reaction was terminated by adding 50 /~1 1 M HCI and the absorbance was read at 490 nm in an ELISA microplate reader (Dynatech, Denkendorf, Germany). c~H~a,t~lctjon

Antigen.Index>-1.2

s

Ac-NH~

5

10

ACSER-ASP-LYS-PRO-ASP-PIET-ALA-GLU-I LE-GLU'~o

~,3 l

15

20

35

40

LYS-PHE-ASP-LYS-SER-LYS-LEU-LYS-LYS-THR25 30 6LU-THR-GLN-GLU-LYS-ASN-PRO-LEU-PRO-SERLYS-GLU-THR-ILE-GLU-GLN-GLU-LYS-GLN-ALAGLY-GLU-SEROH

16

t920

|

31

3

435 36

38

40

4J

42

43

CDOH Fig. 3. Jameson and Wolf plot of thymosin J~4 showing the regions of having a high antigenic index.

134

S. Becker et aL /Journal of Immunological Methods 177 (1994) 131-137

Two tetrapeptides with sequences (ADEFGI, DEFGIK) not related to the native peptide were used as controls. In addition two further negative controls were performed: an epitope mapping with normal rabbit serum, diluted 1/500 in ELISA buffer and an epitope mapping with an antiserum directed against KLH, titre 1/20000, also diluted in ELISA buffer. Both mappings were made to recognise cross reactivities against other serum compounds. The different maximum absorbance between the assays resulted from differences in the incubation times. The results obtained were not corrected for non-specific binding.

OD (490 nm) 2.5

2.0~

1.5

1.0 OD (490 nm) 1.2

1.( 1 0.4

11

21 Pins

31

41 nc

Fig. 5. Epitope mapping of a polyclonai antiserum from rabbit against thymosin /34 (1-14), pins 1-38: hexapeptides 1-6 to 38-43 and a negative control (nc).

0.( 3. Results

0.~

0.;

0.( 1

11

21 Pins

31

41 nc

Fig. 4. Epitope mapping of a polyclonal antiserum from rabbit against thymosin /34, pins 1-38: hexapeptides 1-6 to 38-43 and a negative control (nc).

Theoretical predictions of the antigenic sites (Voelter et al., 1990c) were made using the parameters mentioned in the Introduction (Fig. 3). According to these results, high antigenic sites should be located between the amino acid residues 4-5, 14-15, 24-25 and 29-30. Also, based on 2D-NMR experiments (Zarbock et al., 1990) thymosin /34 in solutions of trifluoroethanol-d3 and hexafluoroisopropyl-p2-alcohol in water adopts a helical structure, covering the segments from residues 4-16 and 30-40. Since the polyclonal antiserum against thymosin /34 exhibited cross-reactivities with the fragment thymosin /34 (1-14), we assumed that an antigenic

135

s. Becket et aL /Journal of Immunological Methods 177 (1994) 131-137

determinant was located in the N-terminal region. Our experimental studies provided evidence for three distinct epitopes, one of which was located at the N-terminus, between the amino acid residues 6-16, as predicted. The two other epitopes found to be present, spanned the amino acid residues 18-26 and 29-37 (Fig. 4). Further epitope mapping, using a polyclonal antiserum against thymosin J~4 (1-14), revealed one epitope (Fig. 5) only. This antigenic determinant was located in the peptide fragments 1-11, indicating a high specificity of the antiserum for the N-terminus of the native peptide and these results were confirmed in replicate experiments. The specificity of the produced antibodies was documented by cross-reactivity studies of the

OD (490 nm) 1.0

0.8

0.6

0.4

OD (490 nm) 1.0

T ,,li,,lll,I,i ,i,,,i,,,,/,ill,,,i,,

0.8

1

11

21

31

Pins

Fig. 7. Epitope mapping of an antiserum directed against KLH as a negative control.

0.6

peptide pins with normal rabbit serum and antiserum against KLH (Figs. 6 and 7). The different maximal absorbances observed resulted from differing incubation times; the results were not corrected for non-specific binding.

0.4

0.2

4. Discussion

0.0 1

11

21 31 Pins Fig. 6. Epitope mapping of a normal rabbit serum as a negative control.

As these results on thymosin J~4 demonstrate, epitope mapping studies with overlapping hexapeptides are an efficient method for the investigations of antigen-antibody interactions. The experimental epitope mapping data are partially consistent with the theoretical predictions of antigenic determinants found in the segments cover-

136

S. Becker et al. /Journal o f lmmunological Methods 177 (1994) 131-137

ing the amino acid residues 6-16 and 29-37 of the thymosin /34 sequence. The discrepancy between theory and experiment found for the epitope segment 18-26 may be caused by one ore more of the following: (i) the hexapeptide structure (on the pins) may not necessarily adopt a conformation identical to that of the native peptide, (ii) by coupling the peptide to KLH with glutardialdehyde, epitopes of the native peptide may be masked and (iii) new epitopes may be generated as a result of conformational changes due to the glutardialdehyde coupling, taking into account the fact that thymosin /34 has about 20 potential coupling sites. Using the antiserum against thymosin /34 (1-14), the epitope mapping experiments identified one single binding site at the N-terminus of thymosin /34, covering the amino acid residues 1-11. Comparing the different possibilities of epitope mapping, the method based on the pin strategy, is the most efficient and detailed approach to the study of antigen-antibody interactions. Contrary to other reports (Tampe et al., 1992) we observed good reproducibility. Nevertheless, this method is suitable only for small peptides up to lengths of about 50 amino acid residues and lacking disulfide bonds. Furthermore interrupted epitopes cannot be recognised, and to avoid misinterpretations resulting from possible conformational changes of the hexapeptides compared to the native peptide, alternative methods for antibody characterisation, such as exchange or deletion of single amino acids, may be used. A further possibility is enzymatic cleavage of peptides or proteins, separation of the fragments with HPLC and using the purified segments for screening. However, these methods are, more tedious compared to the epitope mapping procedure described in this report. The results presented here suggest a range of possibilities for the use of antisera against thymosin /34 (intact) and thymosin /34 (1-14). Because of its cross reactivity with the native peptide the antiserum specific for the fragment thymosin /34 (1-14), can be employed directly for immunohistochemical studies. Furthermore, a combination of both antisera, now makes it possible to develop a sandwich-ELISA with the advan-

tages of high sensitivity and specificity. Moreover, monospecific antibody fractions may be isolated at low cost from the polyclonal antisera by affinity chromatography, using hexapeptides coupled to a solid phase matrix. In conclusion, the production of monospecific antisera, as described in this communication, provides an attractive alternative to the tedious development of monoclonal antibodies, and permits the isolation of different specific antibody populations, from a single polyclonal polyspecific antiserum.

References Atkinson, M.J., Jiippner, H., Niepel, B., Casaretto, M., Zahn, H. and Hesch, R.D. (1982) Characterisation of the binding sites of anti-parathyroid hormone antisera using synthetic parathyroid hormone peptides. J. Immunoassay 3, 31-51. Fischer, J.A., Biswanger, U. and Dietrich, F.M. (1974) Immunological characterisation of antibodies against a glandular extract and the synthetic amino terminal fragments 1-12 and 23-34 and their use in the determination of immunoreactive hormone in human sera. J. Clin. Invest. 54, 1382-94. Geysen, H.M., Meloen, R.H. and Barteling, S.J. (1984) Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid. Proc. Natl. Acad. Sci. USA 81, 3998-4002. Grillon, C., Rieger, K., Bakala, J., Schott, D., Morgat, J.L., Hannappel, E., Voelter, W. and Lefant, M. (1990) FEBS Lett. 274, 30. Hehrmann, R., Nordmeyer, J.P., Mohr, H. and Hensch, R.D. (1980) Human parathyroidhormon: antibody characterisation. J. Immunoassay 1, 151-74. Heinzel, W. and Voelter, W. (1983) Thymushormone und Thymuspeptide. Chem.-Ztg. 105, 291-297. Jameson, B.A. and Wolf, H. (1988) The antigenic index: a novel algorithm for predicting antigenic determinants. Cabios 4, 181. Miheli6, M. and Voelter, W. (1994) Distribution and biological activity of/3-thymosins. Amino Acids 6, 1-13. Miheli6, M., Livaniou, E., H6rger, S., Galic, M., Giebel, W., Lenfant, M. and Voelter, W. (1992) Antibodies against thymosin /34 - Their specificity and use in immunohistochemical studies. Recent Adv. Cell. Mol. Biol. 1, 43-49. Tampe, J., Broszio, P., Manneck, H.E., MiBbichler, A., Blind, E., Miiller, B., Schmidt-Gayk, H. and Armbruster, F.P. (1992) Characterisation of antibodies against human Nterminal parathyroid hormone by epitope mapping. J. Immunoassay 13, 1-13. Visser, T.J., Buurman, C.J. and Birkenhiiger, J.C. (1979) Pro-

S. Becker et al. /Journal of Immunological Methods 177 (1994) 131-137 duction and characterisation of antisera to synthetic 1-34 human parathyroid hormone fragments. Acta Endocrinol. 90, 90-102. Voelter, W., Echner, H., Kalbacher, H., Kapurniotu, A. and Link, P. (1990a) Comparison of solution with solid phase synthesis of thymosin /34. Peptides 1057. Voelter, W., Kalbacher, H., Echner, H., Schmid, B., Treffer, U. and Schr6der, C. (1990b) Liebigs Ann. Chem. 249. Voelter, W., Miheli6, M. and Livaniou, E. (1990c)/3-Thymosin Fragments, their biological activity and use for the development of specific antibodies. Protein StructureFunction. TWEL, New York, pp. 183-197.

137

Van Duijnhoven, H.L.P., Verschuren, M.C.M., Timmer, E.D.J., Vissers, P.M.A.M., Groeneveld, A., Ayoubi, T.A.Y., Van den Ouweland, A.M.W. and Van de Ven, W.J.M. (1991) Application of recombinant DNA technology in epitope mapping and targeting. J. Immunol. Methods 142, 187-188. Zanelli, I.M., Rafferty, B. and Apostolou, B. (1983) Large scale screening programme for selection of antisera for radio immunoassay of human parathyroid hormone. J. Immunoassay 4, 175-206. Zarbock, J., Oschkinat, H., Hannapel, E., Kalbacher, H., Voelter, W. and Holak, T.A. (1990) Biochemistry 29, 7814.