Molecular and Structural Analysis of a Continuous Birch Profilin Epitope Defined by a Monoclonal Antibody
1996; Elsevier BV; Volume: 271; Issue: 47 Linguagem: Inglês
10.1074/jbc.271.47.29915
ISSN1083-351X
AutoresPetra Wiedemann, Klaudia Giehl, Steven C. Almo, А.А. Федоров, Mark E. Girvin, Peter Steinberger, Manfred Rüdiger, Maria Ortner, Manfred J. Sippl, Christiane Dolecek, Dietrich Kraft, Brigitte M. Jockusch, Rudolf Valenta,
Tópico(s)Biochemical and Structural Characterization
ResumoThe interaction of a mouse monoclonal antibody (4A6) and birch profilin, a structurally well conserved actin- and phosphoinositide-binding protein and cross-reactive allergen, was characterized. In contrast to serum IgE from allergic patients, which shows cross-reactivity with most plants, monoclonal antibody 4A6 selectively reacted with tree pollen profilins. Using synthetic overlapping peptides, a continuous hexapeptide epitope was identified. The exchange of a single amino acid (Gln-47 → Glu) within the epitope was found to abolish the binding of monoclonal antibody 4A6 to other plant profilins. The NMR analyses of the birch and the nonreactive timothy grass profilin peptides showed that the loss of binding was not due to major structural differences. Both peptides adopted extended conformations similar to that observed for the epitope in the x-ray crystal structure of the native birch profilin. Binding studies with peptides and birch profilin mutants generated by in vitro mutagenesis demonstrated that the change of Gln-47 to acidic amino acids (e.g. Glu or Asp) led to electrostatic repulsion of monoclonal antibody 4A6. In conclusion the molecular and structural analyses of the interaction of a monoclonal antibody with a continuous peptide epitope, recognized in a conformation similar to that displayed on the native protein, are presented. The interaction of a mouse monoclonal antibody (4A6) and birch profilin, a structurally well conserved actin- and phosphoinositide-binding protein and cross-reactive allergen, was characterized. In contrast to serum IgE from allergic patients, which shows cross-reactivity with most plants, monoclonal antibody 4A6 selectively reacted with tree pollen profilins. Using synthetic overlapping peptides, a continuous hexapeptide epitope was identified. The exchange of a single amino acid (Gln-47 → Glu) within the epitope was found to abolish the binding of monoclonal antibody 4A6 to other plant profilins. The NMR analyses of the birch and the nonreactive timothy grass profilin peptides showed that the loss of binding was not due to major structural differences. Both peptides adopted extended conformations similar to that observed for the epitope in the x-ray crystal structure of the native birch profilin. Binding studies with peptides and birch profilin mutants generated by in vitro mutagenesis demonstrated that the change of Gln-47 to acidic amino acids (e.g. Glu or Asp) led to electrostatic repulsion of monoclonal antibody 4A6. In conclusion the molecular and structural analyses of the interaction of a monoclonal antibody with a continuous peptide epitope, recognized in a conformation similar to that displayed on the native protein, are presented. INTRODUCTIONTo study the mode of the interaction of protein antigens with their antibodies, defined experimental systems are required. In those cases in which crystal structures of antibodies with their corresponding antigen have been determined, it was found that the epitopes (antigenic determinants) belonged to the discontinuous type of epitopes, i.e. several surface loops are involved in the interaction with the corresponding paratope (Amit et al., 2Amit A.G. Mariuzza R.A. Phillips S.E.V. Poljak R.J. Science. 1986; 233: 747-753Crossref PubMed Scopus (976) Google Scholar; Sheriff et al., 44Sheriff S. Silverton E.W. Padlan E.A. Cohen G.H. Smith-Gill S.J. Finzel B.C. Davies D.R. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8075-8079Crossref PubMed Scopus (599) Google Scholar; Padlan et al., 36Padlan E.A. Silverton E.W. Sheriff S. Cohen G.H. Smith-Gill S.J. Davies D.R. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5938-5942Crossref PubMed Scopus (466) Google Scholar; Tulip et al., 55Tulip W.R. Varghese J.N. Webster R.G. Air G.M. Laver W.G. Colman P.M. Cold Spring Harbor Symp. Quant. Biol. 1990; 54: 257-263Crossref Google Scholar; reviewed in Berzofsky, 6Berzofsky J.A. Science. 1985; 229: 932-940Crossref PubMed Scopus (342) Google Scholar; Braden and Poljak, 8Braden C.B. Poljak R.J. FASEB J. 1995; 9: 9-16Crossref PubMed Scopus (183) Google Scholar). In contrast, it has been proposed that epitopes on native proteins consist mainly of short sequence segments of about 6 amino acids that can be mimicked by utilizing synthetic peptides (Green et al., 22Green N. Alexander H. Olson A. Alexander S. Shinnick T.N. Sutcliffe J.G. Lerner R.A. Cell. 1982; 28: 477-487Abstract Full Text PDF PubMed Scopus (511) Google Scholar). Indeed, it was demonstrated that small peptides can elicit antibodies with sequence and structural requirements for binding antigens comparable to antibodies raised against the native protein (Geysen et al., 19Geysen H.M. Barteling S.J. Meloen R.H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 178-182Crossref PubMed Scopus (233) Google Scholar) and that overlapping oligopeptides can be used for epitope analysis (Geysen et al., 20Geysen H.M. Rodda S.J. Mason T.J. Tribbick G. Schoofs P.G. J. Immunol. Methods. 1987; 102: 259-274Crossref PubMed Scopus (719) Google Scholar). Despite these data, the existence of epitopes consisting of small continuous sequence motifs in native proteins has been questioned with the argument that antibodies elicited against peptides might selectively react with denatured, unfolded proteins (Jemmerson and Blankenfeld, 25Jemmerson R. Blankenfeld R. Mol. Immunol. 1989; 26: 301-307Crossref PubMed Scopus (32) Google Scholar). In this context, we studied the interaction of a structurally well defined protein antigen with a monoclonal antibody. We used birch pollen profilin as a model (Valenta et al., 56Valenta R. Duchêne M. Pettenburger K. Sillaber C. Valent P. Bettelheim P. Breitenbach M. Rumpold H. Kraft D. Scheiner O. Science. 1991; 253: 557-560Crossref PubMed Scopus (615) Google Scholar). Profilins are small (14-17 kDa) proteins found in all eukaryotic phyla that bind to actin and to polyphosphoinositol lipids, particularly to phosphatidylinositol 4,5-bisphosphate, and thus may represent a link between the cytoskeleton and signal transduction (Machesky and Pollard, 30Machesky L.M. Pollard T.D. Trends Cell Biol. 1993; 3: 381-385Abstract Full Text PDF PubMed Scopus (145) Google Scholar; Sohn and Goldschmidt-Clermont, 47Sohn R.H. Goldschmidt-Clermont P.J. BioEssays. 1994; 7: 165-172Google Scholar; Drobak et al., 13Drobak B. Watkins P.A.C. Valenta R. Dove S. Lloyd C.W. Staiger C.J. Plant J. 1994; 6: 389-400Crossref Scopus (108) Google Scholar). In addition all profilins bind to poly-L-proline (Tanaka and Shibata, 53Tanaka M. Shibata H. Eur. J. Biochem. 1985; 151: 291-297Crossref PubMed Scopus (144) Google Scholar; Kaiser et al., 27Kaiser D.A. Goldschmidt-Clermont P.J. Levine B.A. Pollard T.D. Cell Motil. Cytoskeleton. 1989; 14: 251-262Crossref PubMed Scopus (80) Google Scholar; Schutt et al., 42Schutt C. Myslik J. Rozycki M. Goonesekere N. Lindberg U. Nature. 1993; 365: 810-816Crossref PubMed Scopus (594) Google Scholar; Björkegren et al., 7Björkegren C. Rozycki M. Schutt C.E. Lindberg U. Karlsson R. FEBS Lett. 1993; 133: 123-126Crossref Scopus (85) Google Scholar; Archer et al., 3Archer S.J. Vinson V.K. Pollard T.D. Torchia D.A. FEBS Lett. 1994; 337: 145-151Crossref PubMed Scopus (57) Google Scholar; Metzler et al., 33Metzler W.J. Bell A.J. Ernst E. Lavoie T.B. Müller L. J. Biol. Chem. 1994; 269: 4620-4625Abstract Full Text PDF PubMed Google Scholar). Recently, the first biologically relevant proline-rich ligand for profilin was identified (Reinhard et al., 37Reinhard M. Giehl K. Abel K. Haffner C. Jarchau T. Hoppe V. Jockusch B.M. Walter U. EMBO J. 1995; 14: 1583-1589Crossref PubMed Scopus (415) Google Scholar).Profilins have also been described as potent allergens (Valenta et al., 56Valenta R. Duchêne M. Pettenburger K. Sillaber C. Valent P. Bettelheim P. Breitenbach M. Rumpold H. Kraft D. Scheiner O. Science. 1991; 253: 557-560Crossref PubMed Scopus (615) Google Scholar; 1992; Vallier et al., 60Vallier P. Dechamp C. Valenta R. Vial O. Deviller P. Clin. Exp. Allergy. 1992; 22: 774-782Crossref PubMed Scopus (151) Google Scholar). IgE antibodies from profilin-allergic patients were shown to cross-react with profilins from different sources, which has led to the designation of profilins as "pan-allergens" (Valenta et al., 57Valenta R. Duchêne M. Ebner C. Valent P. Sillaber C. Deviller P. Ferreira F. Tejkl M. Edelmann H. Kraft D. Scheiner O. J. Exp. Med. 1992; 175: 377-385Crossref PubMed Scopus (577) Google Scholar). In the present study we have analyzed the interaction of birch profilin with a specific mouse monoclonal antibody at the molecular and structural level.mAb 1The abbreviations used are: mAbmonoclonal antibodyHPLChigh pressure liquid chromatographyELISAenzyme-linked immunosorbent assayCDRcomplementary determining regionPCRpolymerase chain reactionPBSphosphate-buffered salineTOCSYtotal correlation spectroscopyROESYrotating frame Overhauser effect spectroscopy. 4A6 bound to a continuous hexapeptide epitope that, according to the comparison of the peptide NMR analysis and the crystal structure of birch profilin, formed a similar conformation as in the native protein. Gln-47 was determined as the crucial amino acid for the contact with mAb 4A6 using structural data, peptide variants, and protein mutants.RESULTS AND DISCUSSIONThe interaction of birch pollen profilin and a specific mouse monoclonal antibody, designated 4A6, was investigated. Birch profilin (Valenta et al., 56Valenta R. Duchêne M. Pettenburger K. Sillaber C. Valent P. Bettelheim P. Breitenbach M. Rumpold H. Kraft D. Scheiner O. Science. 1991; 253: 557-560Crossref PubMed Scopus (615) Google Scholar; 1992; 1993) was chosen as a model for antigen-antibody interactions for two reasons. First, although they display only modest sequence homology, profilins are structurally well conserved eukaryotic proteins, which may be due to their conserved function as actin-binding proteins (Almo et al., 1Almo S.C. Pollard T.D. Way M. Lattman E.E. J. Mol. Biol. 1994; 236: 950-952Crossref PubMed Scopus (27) Google Scholar; Fedorov et al., 15Fedorov A.A. Pollard T.D. Almo S.C. J. Mol. Biol. 1994; 241: 480-482Crossref PubMed Scopus (47) Google Scholar). Indeed it could be shown that despite a low degree of sequence similarity, profilin and actin from different species could interact in vitro as well as in vivo (Valenta et al., 58Valenta R. Ferreira F. Grote M. Swoboda I. Vrtala S. Duchêne M. Deviller P. Meagher R.B. McKinney E. Heberle-Bors E. Kraft D. Scheiner O. J. Biol. Chem. 1993; 268: 22777-22781Abstract Full Text PDF PubMed Google Scholar; Giehl et al., 21Giehl C. Valenta R. Rothkegel M. Ronsiek M. Mannherz H.-G. Jockusch B. Eur. J. Biochem. 1994; 226: 681-689Crossref PubMed Scopus (64) Google Scholar; Staiger et al., 49Staiger C.J. Yuan M. Valenta R. Shaw P.J. Warn R.M. Lloyd C.W. Curr. Biol. 1994; 4: 215-219Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar; Rothkegel et al., 39Rothkegel M. Mayboroda O. Rohde M. Wucherpfennig C. Valenta R. Jockusch B.M. J. Cell Sci. 1996; 109: 83-90PubMed Google Scholar). Additionally, profilins are potent allergens that induce cross-reactive IgE antibodies in about 20% of allergic patients (Valenta et al., 56Valenta R. Duchêne M. Pettenburger K. Sillaber C. Valent P. Bettelheim P. Breitenbach M. Rumpold H. Kraft D. Scheiner O. Science. 1991; 253: 557-560Crossref PubMed Scopus (615) Google Scholar).mAb 4A6 consists of an IgG1 heavy chain and a κ light chain. The deduced amino acid sequence of the 4A6 amino-terminal heavy chain fragment and its corresponding light chain are shown in Fig. 1. In the CDRs of the light chain two acidic amino acids were found, whereas in the CDRs of the heavy chain five acidic amino acids were observed. Despite a high degree of sequence identity of approximately 80% among profilins from higher plants, mAb 4A6 was able to discriminate between tree pollen profilins and other plant profilins (Fig. 2). The 4A6 epitope was mapped using synthetic dodecapeptides that spanned the deduced amino acid sequence of birch profilin by 10 amino acids of overlap. mAb 4A6 bound strongly to peptides (amino acids 38-49 and amino acids 40-51) of birch profilin, whereas peptides (amino acids 36-47 and amino acids 42-53) reacted more weakly. All peptides reacting with 4A6 shared the 6-amino acid sequence motif PQFKPQ. This sequence motif was compared with the relevant region in other plant profilins (Staiger et al., 48Staiger C.J. Goodbody K.C. Hussey P.J. Valenta R. Drobak B.K. Lloyd C.W. Plant J. 1993; 4: 631-641Crossref PubMed Scopus (129) Google Scholar; Valenta et al., 59Valenta R. Ball T. Vrtala S. Duchêne M. Kraft D. Scheiner O. Biochem. Biophys. Res. Commun. 1994; 199: 106-118Crossref PubMed Scopus (78) Google Scholar; Rihs et al., 38Rihs H.-P. Rozynek P. May-Taube K. Welticke B. Baur X. Int. Arch. Allergy Immunol. 1994; 105: 190-194Crossref PubMed Scopus (40) Google Scholar; Mittermann et al., 34Mittermann I. Swoboda I. Pierson E. Eller N. Kraft D. Valenta R. Heberle-Bors E. Plant Mol. Biol. 1995; 27: 137-146Crossref PubMed Scopus (81) Google Scholar). The only consistent sequence difference between birch profilin and the other plant profilins was seen in the last position of the hexapeptide. Here, only birch profilin contained Gln-47 instead of Glu.Fig. 2Reactivity of mAb 4A6 and anti-plant profilin antisera with nitrocellulose-blotted profilins from different plant species. Protein extracts were prepared from pollens of different plant species, comprising dicotyledonic plants (birch, Betula verrucosa; alder, Alnus glutinosa; mugwort, Artemisia vulgaris; tobacco, Nicotiana tabacum) and monocotyledonic plants (timothy grass, Phleum pratense; maize, Zea mais; wheat, Triticum sativum), separated by SDS-polyacrylamide gel electrophoresis, and blotted to nitrocellulose. Nitrocellulose strips were then incubated with the mouse monoclonal anti-birch profilin antibody (4A6), a mouse monoclonal antibody without specificity for profilins (mK), a rabbit antiserum raised against celery root profilin (RP1), a rabbit antiserum raised against recombinant birch profilin (RP2), a rabbit antiserum raised against the birch profilin carboxyl terminus (RP3), and a normal rabbit serum (nrs). Antibodies bound to profilin at 14 kDa were detected with a 125I-labeled sheep anti-mouse and donkey anti-rabbit antiserum, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The difference in binding of mAb 4A6 to different plant profilins was further investigated at the epitope level using peptides corresponding to the birch, timothy grass, and tobacco epitope. Peptides comprising 14 amino acids of the plant profilins were probed for binding to mAb 4A6 by dot blotting (Fig. 3). 4A6 did not bind to the timothy grass and tobacco peptides but reacted with the birch epitope peptides synthesized as 14-mer, 12-mer, and hexapeptide. The binding intensity decreased with the length of the peptides. Thus, as the minimal epitope for mAb 4A6, the birch hexapeptide PQFKPQ was identified, which differed from the other plant profilin peptides by a single amino acid (Gln-47 → Glu).Fig. 3Reactivity of mAb 4A6 with peptides from different plant profilins tested by dot blotting. In lane 1 peptides were tested with a monoclonal antibody without specificity for birch profilin, and in lane 2 mAb 4A6 was used. Approximately 100 ng of each peptide were dotted to the nitrocellulose in the order shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To compare the affinity of recombinant birch profilin with a synthetic peptide epitope spanning amino acids 36-51, competitive ELISA studies were performed (Fig. 4). Purified recombinant birch profilin was coated to ELISA plates and probed with mAb 4A6 that was preincubated either with purified recombinant birch profilin or the synthetic birch profilin peptide BP36/51. The concentration for a 50% competition with recombinant birch profilin was determined to be 1.2 × 10−7M for recombinant birch profilin and 5 × 10−8M for the peptide BP36/51, when 50 ng of purified mAb 4A6 were used per well. Thus, the peptide BP36/51 displayed a slightly higher affinity for mAb 4A6 than the complete recombinant birch profilin.Fig. 4ELISA competition assay. ELISA plates were coated with 100 ng of recombinant birch profilin/well. Purified mAb 4A6 was allowed to bind to recombinant birch profilin using increasing concentrations (x axis) of recombinant birch profilin (∘) or peptide BP 36/51 (▴) as competitor for preincubation. The extinction is displayed as relative absorbance at 490 nm on the y axis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Hence, the 4A6 epitope represents a continuous epitope, a term coined for peptide epitopes consisting of short sequence motifs (Berzofsky, 6Berzofsky J.A. Science. 1985; 229: 932-940Crossref PubMed Scopus (342) Google Scholar). Although continuous epitopes have been reported for a number of antigens, and antibodies were described that bound with comparable affinity to a peptide epitope and the complete native protein (Navon et al., 35Navon A. Schulze A.J. Guillou Y. Zylinski C.A. Baleux F. Expert-Bezancon N. Friguet B. Djavadi-Ohaniance L. Goldberg M.E. J. Biol. Chem. 1995; 270: 4255-4261Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar; Fernandez et al., 16Fernandez J.A. Villoutreix B.O. Hackeng T.M. Griffin J.H. Bouma B.N. Biochemistry. 1994; 33: 11073-11078Crossref PubMed Scopus (21) Google Scholar), the physiological role of continuous epitopes has been questioned (Laver et al., 29Laver W.G. Air G.M. Webster R.G. Smith-Gill S.J. Cell. 1990; 61: 553-556Abstract Full Text PDF PubMed Scopus (427) Google Scholar).Crystallographic analyses of antigen-antibody complexes of intact proteins demonstrated that binding predominantly involves conformational epitopes, which are assembled from multiple peptide segments separated in the primary sequence (reviewed in Braden and Poljak, 8Braden C.B. Poljak R.J. FASEB J. 1995; 9: 9-16Crossref PubMed Scopus (183) Google Scholar). Such conformational epitopes have been described for other birch pollen allergens. A calcium-binding birch pollen allergen, Bet v 3, contained an epitope that was sensitive to depletion of calcium and denaturation (Seiberler et al., 43Seiberler S. Scheiner O. Kraft D. Lonsdale D. Valenta R. EMBO J. 1994; 15: 3481-3486Crossref Scopus (118) Google Scholar). IgE epitopes of the major birch pollen allergen Bet v 1 (Breiteneder et al., 9Breiteneder H. Pettenburger K. Bito A. Valenta R. Kraft D. Rumpold H. Scheiner O. Breitenbach M. EMBO J. 1989; 8: 1935-1938Crossref PubMed Scopus (648) Google Scholar) could not be determined with overlapping peptides, and protein fragments did not demonstrate IgE antibody binding. 3S. Vrtala, K. Hirtenlehner, L. Vangelista, A. Pastore, H.-G. Eichler, W. R. Sperr, P. Valent, C. Ebner, D. Kraft, and R. Valenta, submitted for publication.In order to obtain information whether the different binding of mAb 4A6 to the plant peptides might be due to conformational differences, the protein backbone conformations for the birch, maize, timothy grass, tobacco, and wheat peptides were calculated from the data base with the Boltzmann device (Sippl, 45Sippl M.J. J. Mol. Biol. 1990; 213: 859-883Crossref PubMed Scopus (950) Google Scholar), revealing a rather similar structure for the different peptides (data not shown). The prediction was confirmed by NMR analysis of the birch (SFPQFKPQEITG) and timothy (SFPQFKPEEITG) peptide. Both peptides showed extended conformation in solution (Fig. 5). The alignment of the ensemble of NMR structures calculated for the birch P3-Q8 peptide segment gave a consistent set of structures, whereas the timothy P3-E8 peptide segment displayed more variability, due to fewer and weaker nuclear Overhauser effects.Fig. 5Superimposition of the 10 best NMR structures of the QE (birch, left) and EE (timothy, right) peptides. The Cα traces, along with proline side chains, are shown for the residues corresponding to positions 42-47 of the birch profilin sequence. The structures were aligned to minimize the root mean squared deviations between the positions of equivalent backbone atoms.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The x-ray structure of birch profilin, determined at 2.4-Å resolution,2 also showed that the 4A6 epitope adopted an extended conformation in the native birch profilin molecule. When the peptide was considered in the context of the folded protein (Table I) a significant burial of surface area is seen for only two amino acids. Pro-42 and Phe-44 have a large buried surface area due to extensive packing in the hydrophobic core of birch pollen profilin. In contrast, Gln-43, Lys-45, Pro-46, and Gln-47 are positioned at the surface of the folded molecule and are thus accessible to the solvent. Fig. 6 shows the superposition of the two peptide epitope structures as determined by NMR with the observed crystal structure for residues 42-47 from birch pollen profilin. Although the fits were not complete (root mean sequence of 1.38 and 1.6 Å on backbone atoms of the birch and timothy peptide, respectively), the extended conformation of both peptides suggested that they may readily conform to the appropriate conformation required for antibody binding.Table I.Solvent accessibility of the 4A6 epitope in the protein background and in isolationAmino acidAccessibility in intact proteinAccessibility in isolated peptideBuried surfaceaBuried surface area is calculated as the difference in calculated accessibility of residues in the isolated peptide and the peptide in the intact protein (i.e. accessible surface in isolated peptide − accessible surface area in protein).BuriedbPercent buried is calculated as (accessible surface in isolated peptide − accessible surface area in protein)/(accessible surface area in protein).Å2Å2Å2%Pro-424018114178Gln-431461763017Phe-442316814586Lys-451241755129Pro-46871253830Gln-471111766537a Buried surface area is calculated as the difference in calculated accessibility of residues in the isolated peptide and the peptide in the intact protein (i.e. accessible surface in isolated peptide − accessible surface area in protein).b Percent buried is calculated as (accessible surface in isolated peptide − accessible surface area in protein)/(accessible surface area in protein). Open table in a new tab Fig. 6Stereo view of the alignment of the QE (birch, red) and EE (timothy, green) peptide NMR structures with the corresponding loop from the birch profilin crystal structure (white). The C′, Cα, and N backbone atoms of the peptides and profilin were used in the alignment.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Based on the assumption that the free birch peptide and the epitope within the native molecule make the same contacts with mAb 4A6, two models of interaction were considered. One possibility was that 4A6 binds to the epitope without requiring a significant change in the epitope conformation. In this model, Gln-43, Lys-45, Pro-46, and Gln-47 would make extensive contacts with the CDRs, whereas Pro-42 and Phe-44 would not contact the CDRs. A second model involves a conformational change of the epitope upon binding to 4A6 such that residues with low accessibility in the native protein would make significant contributions to the binding interface. However, if Phe-44 was involved in complex formation, it would have to leave the hydrophobic core of the protein. Considering the energetic cost of such a rearrangement, this possibility seemed unlikely.The comparison of the NMR structure of the birch peptide with the timothy peptide containing the Gln-47 → Glu exchange showed that both peptides have an extended conformation that could be superimposed to the 4A6 epitope deduced from the crystal structure of birch profilin. These data indicated that the lack of cross-reactivity of mAb 4A6 with other plant profilins was not due to a local conformational change of this epitope due to the Gln-47 → Glu exchange. Binding tests with mutant peptides (Fig. 7) demonstrated that a change of Phe-44 to Tyr-44 did not affect the 4A6 binding. Gln-47 could be exchanged to Asn-47 without altering 4A6 reactivity. However, the substitution of Gln-47 by acidic amino acids such as Glu-47 or Asp-47 abolished binding of 4A6 completely.Fig. 7Reactivity of mAb 4A6 with mutant birch profilin peptides. mAb 4A6 was tested with dot-blotted mutant peptides. The sequences and order of the peptides are displayed.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The peptide binding data could be reproduced using recombinant birch profilin mutants (Fig. 8). Birch profilin mutants Phe-44 → Tyr and Gln-47 → Asn were bound by mAb 4A6, whereas the Gln-47 → Glu mutant was not recognized. A band of approximately 28 kDa observed in the Phe-44 → Tyr mutant preparation was recognized by the antibodies and therefore most likely represented a dimer. Antibodies with specificity for other epitopes (serum IgE from a birch profilin allergic individual or a rabbit antiserum raised against the birch profilin carboxyl terminus RP3) reacted with all three mutant proteins. In addition all birch profilin mutants could be purified by poly-L-proline affinity chromatography, indicating correct folding and functional activity of the molecules (Vrtala et al., 62Vrtala S. Wiedemann P. Mittermann I. Eichler H.G. Sperr W.R. Valent P. Kraft D. Valenta R. Biochem. Biophys. Res. Commun. 1996; (in press)PubMed Google Scholar).Fig. 8Reactivity of nitrocellulose-blotted recombinant birch profilin (wild type) and birch profilin mutants with antibodies. E. coli extracts containing approximately 1 μg/cm gel recombinant birch profilin wild type (P) and mutagenized birch profilins (clone 4, Phe-44 → Tyr; clone 25, Gln-47 → Glu; clone 35, Gln-47 → Asn) were separated by SDS-polyacrylamide gel electrophoresis and blotted onto nitrocellulose. Nitrocelluloses were probed with serum IgE from a profilin-allergic patient, mAb 4A6, and a rabbit anti-birch carboxyl terminus antiserum.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The binding experiments with peptides and birch profilin mutants together supported the first model of interaction, which involves Gln-43, Lys-45, Pro-46, and Gln-47 as direct contact sites of birch profilin with mAb 4A6. It is further assumed that changes of Gln-47 to structurally similar acidic amino acids such as Glu or Asp abolished binding, most likely as a consequence of electrostatic repulsion caused by acidic amino acid residues present in the CDRs of mAb 4A6. This hypothesis was corroborated by the fact that changes of Gln-47 to Asn, an amino acid of similar structure and functionality, did not abolish binding of mAb 4A6.In conclusion we have characterized a monoclonal antibody specific for a potent allergen that is an important component of the plant cytoskeleton. A continuous hexapeptide motif was identified as the minimal epitope and studied at the molecular and structural level. It was demonstrated that the natural immune response toward protein antigens can result in the production of peptide-directed antibodies that derive substantial binding energy from linear epitopes. These analyses may contribute to the general concepts on epitope-paratope interactions. INTRODUCTIONTo study the mode of the interaction of protein antigens with their antibodies, defined experimental systems are required. In those cases in which crystal structures of antibodies with their corresponding antigen have been determined, it was found that the epitopes (antigenic determinants) belonged to the discontinuous type of epitopes, i.e. several surface loops are involved in the interaction with the corresponding paratope (Amit et al., 2Amit A.G. Mariuzza R.A. Phillips S.E.V. Poljak R.J. Science. 1986; 233: 747-753Crossref PubMed Scopus (976) Google Scholar; Sheriff et al., 44Sheriff S. Silverton E.W. Padlan E.A. Cohen G.H. Smith-Gill S.J. Finzel B.C. Davies D.R. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8075-8079Crossref PubMed Scopus (599) Google Scholar; Padlan et al., 36Padlan E.A. Silverton E.W. Sheriff S. Cohen G.H. Smith-Gill S.J. Davies D.R. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5938-5942Crossref PubMed Scopus (466) Google Scholar; Tulip et al., 55Tulip W.R. Varghese J.N. Webster R.G. Air G.M. Laver W.G. Colman P.M. Cold Spring Harbor Symp. Quant. Biol. 1990; 54: 257-263Crossref Google Scholar; reviewed in Berzofsky, 6Berzofsky J.A. Science. 1985; 229: 932-940Crossref PubMed Scopus (342) Google Scholar; Braden and Poljak, 8Braden C.B. Poljak R.J. FASEB J. 1995; 9: 9-16Crossref PubMed Scopus (183) Google Scholar). In contrast, it has been proposed that epitopes on native proteins consist mainly of short sequence segments of about 6 amino acids that can be mimicked by utilizing synthetic peptides (Green et al., 22Green N. Alexander H. Olson A. Alexander S. Shinnick T.N. Sutcliffe J.G. Lerner R.A. Cell. 1982; 28: 477-487Abstract Full Text PDF PubMed Scopus (511) Google Scholar). Indeed, it was demonstrated that small peptides can elicit antibodies with sequence and structural requirements for binding antigens comparable to antibodies raised against the native protein (Geysen et al., 19Geysen H.M. Barteling S.J. Meloen R.H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 178-182Crossref PubMed Scopus (233) Google Scholar) and that overlapping oligopeptides can be used for epitope analysis (Geysen et al., 20Geysen H.M. Rodda S.J. Mason T.J. Tribbick G. Schoofs P.G. J. Immunol. Methods. 1987; 102: 259-274Crossref PubMed Scopus (719) Google Scholar). Despite these data, the existence of epitopes consisting of small continuous sequence motifs in native proteins has been questioned with the argument that antibodies elicited against peptides might selectively react with denatured, unfolded proteins (Jemmerson and Blankenfeld, 25Jemmerson R. Blankenfeld R. Mol. Immunol. 1989; 26: 301-307Crossref PubMed Scopus (32) Google Scholar). In this context, we studied the interaction of a structurally well defined protein antigen with a monoclonal antibody. We used birch pollen profilin as a model (Valenta et al., 56Valenta R. Duchêne M. Pettenburger K. Sillaber C. Valent P. Bettelheim P. Breitenbach M. Rumpold H. Kraft D. Scheiner O. Science. 1991; 253: 557-560Crossref PubMed Scopus (615) Google Scholar). 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In the present study we have analyzed the interaction of birch profilin with a specific mouse monoclonal antibody at the molecular and structural level.mAb 1The abbreviations used are: mAbmonoclonal antibodyHPLChigh pressure liquid chromatographyELISAenzyme-linked immunosorbent assayCDRcomplementary determining regionPCRpolymerase chain reactionPBSphosphate-buffered salineTOCSYtotal correlation spectroscopyROESYrotating frame Overhauser effect spectroscopy. 4A6 bound to a continuous hexapeptide epitope that, according to the comparison of the peptide NMR analysis and the crystal structure of birch profilin, formed a similar conformation as in the native protein. Gln-47 was determined as the crucial amino acid for the contact with mAb 4A6 using structural data, peptide variants, and protein mutants.
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