J. Mol. Biol. (1992) 225, 577-583
Precise Epitope Mapping Using a Filamentous Phage Epitope Library Charles W. Stephen and David P. Lane Cell Transformation Research Group CRC Laboratories, University of Dundee Dundee DDl 4HN, Scotland (Received 16 January
1992; accepted 21 February
Many naturally occurring point mutations in the p53 gene lead to a proportion of the encoded protein molecules adopting a distinct, “mutant” conformation characterized by exposure of a normally cryptic epitope recognized by the monoclonal antibody PAb240. Here the PAb240 epitope is defined using a filamentous phage epitope library. The hexapeptides displayed by the PAb240-binding phage isolated from the library were all highly related and allowed both direct localization of the epitope and prediction of a specific interaction between PAb240 and Xenopus TFIIIA. This study demonstrates for the first’ time the power of phage epitope libraries in the precise definition of previously unmapped epitopes. Identification of the PAb240 epitope precisely defines a region of the p53 molecule structurally altered by the mutation-induced conformational shift. Keywords:
~53; PAb240; TFIIIA;
Somatic point’ mutations in the p53 gene are found in a wide range of human malignancies, and germ line mutations in ~53 are also responsible for at least one inherited cancer susceptibility syndrome (for reviews, see Lane & Benchimol, 1990; Levine et al., 1991). Over 200 naturally occurring mutations in p53 have now been catalogued (Hollstein et al., 1991). They are clustered in the central region of the molecule (exons 4 to 9) and are often located at evolutionarily conserved residues (Soussiet al., 1990). Many of these mutations lead to a variable proportion of the encoded protein molecules adopting a distinct “mutant” conformation (Finlay et al., 1988; Sturzbecher et al., 1988a; Milner & Cook, 1986; Gannon et al., 1990; Bartek et al., 1990; Rodrigues et al., 1990). Molecules in the mutant conformation are distinguished from wildtype molecules by lack of expression of the denaturation-sensitive epitopes recognized by the monoclonal antibodies PAb246 (Yewdell et al., 1986) and PAb1620 (Ball et al., 1984) and the appearance of a new, normally cryptic epitope recognized by the monoclonal antibody PAb240 (Gannon et al., 1990). Molecules in the PAb240-reactive conformation are often found to be associated with the cellular heat shock protein hsc70 (Finlay of al.. 1988; Sturzbecher et al., 1988a;
filamentous phage; epitope library
Hinds et al., 1990). This association is not observed with protein in the wild-type conformation. The various naturally occurring p53 mutations result in proteins possessing different biological activities (Levine et al., 1991). Differences have been observed in assays for cellular transformation (Halevy et al., 1990; Hinds et al., 1990), inhibition of viral DNA replication (Braithwaite et al., 1987; Sturzbecher et al., 19886; Friedman et aZ., 1990), T antigen binding (Jenkins et al., 1988) and transcriptional activation (Fields & Jang, 1990; Raycroft et al., 1990). The activities displayed in these assays can often be correlated with the strength of reaction of the p53 protein with PAb240 (i.e. with the proportion of molecules adopting the mutant conformation). Mutant ~53 proteins more strongly reactive with PAb240 appear to be more potent in transformation assays than those that are only weakly reactive. For example, human p53 genes containing a His175 mutation, which produce protein strongly reactive with PAb240, have been found to be three- to tenfold more efficient in transformation assays than genes with a His273 mutation that produce p53 protein that is only weakly reactive with PAb240 (Halevy et al., 1990; Hinds et al., 1990; Bartek et al., 1990; Rodrigues et al., 1990). In assays for transcriptional activation,
C. W. Stephen
wild-type murine p53 and human p53 with a His273 mutation are transactivating, whereas a murine ~53 with a linker insertion at residue 215, known to be strongly reactive with PAb240, has no activity (Fields & Jang, 1990; Raycroft et al., 1990). Wild-type murine p53 binds simian virus 40 (SV40) large T antigen, whereas strongly PAb240-positive ~53 proteins do not (Jenkins et al., 1988; Gannon et al., 1990). A further striking correlation between the conformational shift and p53 function has been found in studies of the temperature-sensitive Va1135 mutation in murine ~53 (Michalovitz et al., 1990). At 39°C the encoded protein is dominantly transforming and present in the cytoplasm, At 32°C the protein moves to the nucleus where it acts as a transformation suppressor. The shift from 39°C to 32 “C is accompanied by a decreasein the proportion of PAb240-positive molecules and an increase in the proportion of PAb246-positive molecules (Gannon & Lane, 1991; Martinez et al., 1991; Ginsberg et al., 1991; Milner & Medcalf, 1980). To further understand the nature of the conformational change at the molecular level we needed to map the PAb240 epitope precisely. Using bacterial expression systems the epitope had been mapped to amino acids 156 to 214 of murine p53 (Gannon et al., 1990). The antibody has also been found to bind rodent, primate and chicken p53 but not to interact with Xenopus ~53. We now describe the successful use of a filamentous phage epitope library to localize the epitope precisely. The construction of several phage epitope libraries has been described (Scott & Smith, 1990; Devlin et al., 1990; Cwirla et al., 1990; Felici et al., 1991). These libraries have been constructed using Ff filamentous phage (M13/fd)derived phage or phagemid vectors. They exploit the ability to fuse foreign peptide sequencesto the N terminus of either the phage gene III coat protein (~111) or the phage gene VIII coat protein (pVII1) (Smith, 1985; Parmley 6 Smith, 1988; Greenwood et al., 1991). The foreign peptide sequences are displayed at the surface of the virus particles and are available for antibody binding. The library used in this study was constructed by Jamie Scott and George Smith (University of Missouri, Columbia) using the phage fd-derived vector, fUSE5, and consists of 2 x lo8 independent phage clones each expressing a random hexapeptide fused to pITI. The library is calculated to represent 69% of the 6.4 x lo7 possible hexapeptides (Scott & Smith, 1990). (a) Isolation
and characterization binding phage
Phage reactive with PAb240 were selected from the epitope library using the streptavidin-biotin based “biopanning” procedure described by Parmley & Smith (1988). A library sample containing 68 x 10’ infectious phage particles was subjected to three rounds of selection and amplification using an antibody concentration of 61 mg/ml for the first round and 61 pug/ml for subsequent
and D. P. Lane
rounds. Phage was purified from 42 independent clones obtained after the third selection and assayed for PAb240 binding by antibody-capture ELISAP. Wells were coated with approximately 2 x 10” phage particles by incubation overnight at 4°C and, following blocking, were reacted with purified PAb240 at 1 pg/ml overnight at, 4°C. Bound PAb240 was detected as described in the legend to Figure 3(c). A wild-type phage control gave a background signal of 9080. Phage clones giving a signal above 0230 (-65%) were considered positive. The average signal obtained from the positive clones was 0452; the maximal signal 0.757. DNA was purified from a random selection of positive clones and t,hr gene III insert sequenced directly. All phage clones sequenced were found to contain one of four insert sequences(Fig. l(a)), implying isolation of at least four independent phage from the original library sample. The insert sequencesencoded highly related hexapeptides, all four sharing a common RHSV tetrapeptide core sequence. The C-terminal residue of the encoded hexapeptide was either valinr or isoleucine. The first residue of the hexapeptide showed more variation, but a selection for large; hydrophobic residues was noted. (b) An RHSV
is the PAb240
Examination of the p53 amino acid sequence revealed the presenceof an RHSVV motif conserved between human, mouse and chicken ~53, which all bind PAb240 (Fig. l(b)). PAb240 does not bind t,o Xenopus ~53, which has the sequence RHSVC at) this site. The epitope localization was confirmed hy examining a human ~53 cDNA 3’ deletion series expressed in Escherichia coli. React,ivity of expressed protein with PAb240 was examined by immunoblotting. Protein fragments terminating at or beyond residue 218 were reactive whereas those terminating at or before amino acid 214 were unreactive (data not shown). This was consistent with the RHSVV motif (residues 213 to 217) being the site of the PAb240 epitope. Missenseand nonsense mutations in the RHSV\: motif have both been found in human tumours (Hollstein et al., 1991). The Burkitt’s lymphoma cell line, Raji, has been shown to possess a point mutation in one allele of t,he p53 gene leading to an Arg+Gln substitution at position 213. The other allele is wild-type (Farrell et al., 1991). It was predicted that, if the epitope identification was correct, this mutation would abolish PAb240 binding. Due to heterozygosity at the polymorphic amino acid 72, it was possible to distinguish t,he protein products of the two alleles due to their differential mobility on SDS/PAGE (Matlashewski et aE., 1987). Following Western blotting, filters were probed with PAb421, a monoclonal antibody recognizing a denaturation-resistant, epitopr in t’he C t Abbreviation immunosorbent
used: KLISA. array.
579 No. of independent clones sequence
(0) PAb 240 reactivity Phage sequence Phoge sequence Phage sequence Phage sequence
A 9 C D
Mouse Chicken Xenopus
p53 p53 p53
Xenopus TFIIIA Mutant mouse p53
R H 5L R H 5WRHSVVlO 5 L R H
212 F 209 198
187 270 209
F K G E F
SV S V
+ + +
H H H H
S S S S
V V V V
V V V C
217 214 203 192
R H Cl H
R R R R
( b) Figure 1. (a) Nucleotide sequences and encoded amino acid sequences of the gene III inserts in the 4 isolated PAbZ40-reactive phage types. Single-stranded DNA was isolated from purified phage and the gene III insert sequenced using a Sequenase Version 2.0 kit (United States Biochemical) according to the manufacturer’s instructions. (b) Alignment of amino acid sequences examined for PAb240 reactivity and positions of first and last residues of each hexapeptide within the sequences of the proteins containing them. p53 amino acid sequences were obtained from Soussi et al. (1990) and X. Zaevis TFIIIA amino acid sequence from Ginsberg el al. (1984).
terminus of human and murine p53 (Harlow et al., 1981), and with PAb240 (Fig. 2(a)). PAb421 (lane 1) identified two bands corresponding to the products of the two p53 alleles. PAb240 (lane 2) recognized only the lower (wild-type) p53 band and not the upper (mutant) p53 band. A cross-reacting, approximately 70,000 MT band was also recognized by PAb240. This protein has been detected in other cell lines and appears to be unrelated to ~53. The Gln213 mutation present in the Raji cell line was further shown to result in loss of antibody reactivity by introduction of the equivalent mutation (Arg210 + Gln) into a full-length murine p53 cDNA expressed in E. coli. Extracts of cells expressing the mutant p53 protein were examined by immunoblotting with PAb248, PAb421 and PAb240 (Fig. 2(b)). PAb248 is a monoclonal antibody recognizing a denaturation-resistant epitope in the N terminus of murine p53 (Yewdell et al., 1986). The mutant protein was recognized by PAb248 (lane 1) and PAb421 (lane 5), but not by PAb240 (lane 3). Thus, a full length protein was expressed but the presence of a single substitution at residue 210 abolished PAb240 binding.
(c) The epitope mapping predicts a cross reaction with TFIIIA It was predicted that other proteins containing the sequence RHSVV would also be reactive with PAb240. A computer search of the Swiss Prot protein sequence database revealed the presence of an RHSVV motif in the Xenopus laevis transcription factor TFIIIA between residues 271 and 275 (Ginsberg et al., 1984). To investigate the predicted interaction between PAb240 and X. laevis TFIIIA, X. laevis oocyte extract was examined by immunoblotting (Fig. 3(a)). PAb240 (lane 1) recognized a single major band that comigrated with a band recognized by the anti-TFIIIA monoclonal antibody, 06C5 (lane 2). PAb421 (lane 3) did not recognize a band in this region. To confirm that the band recognized by PAb240 was indeed TFIIIA, purified X. laevis TFIIIA, in the form of 7 S particles, was examined by immunoblotting (Fig. 3(b)). The protein band was recognized by PAb240 (lane 2) and 06C5 (lane 3) but not by control antibodies PAb248 (lane 1) and PAb421 (lane 4). The interaction
C. W. Stephen and ll. Y. Lane (0)
MC (K I IO-‘)
(K I IO-‘)
3, ‘5” &:s ,/ ’
Figure 2. (a) Immunoblots of Raji whole cell extract. Lane 1 was probed with PAb421 and lane 2 with PAb240. Arrows indicate the products of the p53 alleles. Proteins were separated by SDS/PAGE and transferred electrophoretically to mtrocellulose (Schleicher and Schuell). Following blocking, blots were incubated with neat tissue culture supernatant or purified antibody at 10 pg/ml (2 h: room temperature), washed, and incubated with alkaline phosphatase-conjugated rabbit anti-mouse immunoglobulins (Dako) diluted 1 : 1000 (1 h, room temperature). Following washing, bound antibody was visualized using bromochloroindoyl phosphate/nitro blue tetrazolium. (b) Immunoblots of whole cell extract from bacterial cultures expressing Arg210 + Gln mutant murine ~53 (lanes 1, 3 and 5) or wild-type murine ~53 (lanes 2, 4 and 6). Lanes 1 and 2 were probed with PAb248, lanes 3 and 4 with P.4b240. and lanes 5 and 6 with PAb421. The arrow indicates t,he posit,ion of t.he p53 protein. :Z pT7-i based plasmid construct expressing full-1engt.h. wild-type murine p53 ((1. .4. Midgley & D. P. Lane. unpublished results) under control of the bacteriophage TT @IO promoter (Tabor & Richardson, 1985) was modified b> polymerase chain reaction mutagenesis as described b,v Erlich (1989). A mutant oligonucleotide was used to convert codon 210 from CGC to CAG, altering the encoded amino acid from arginine to glutamine. Modified and unmodified plasmids were transferred to E. coli RT,21(DE3) and protein expression induced by the addition of isopropyl-thio-/-n-galactoside to 1 mM. The immunoblotting procedure was as described for (a) except that the immobilized primary antibody was detected with horseradish peroxidase-conjugated rabbit anti-rnousla immunoglobulins (Dako) diluted 1 : 1000 (I h. room temperature) and visualized using the ECL Western blotting system (Amersham) according to the manufacturer’s instructions.
between PAb240 and the purified TFIIIA was further examined by a quantitative, antibodycapture ELISA (Fig. 3(c)). Similar amounts of PAb240 and 06C5 were bound by TFIIIA at each concentration examined while control antibodies PAb421 and PAb419 gave consistent background signals at each TFIIIA concentration.
In previous studies, phage clones were isolated from epitope libraries using model tnonoclonal antibodies previously known t,o react with short. defined peptides in solution. The reactive phage clones were found to display sequences with homology to the known peptide ligands (Scott & Smith, ct al.. 1991). This is 1990: Cwirla et al.. 1990: F&i the first isolation of reactive phage using a monoclonal antibody raised against a full-length protein and immunogen reactive with a previously unmapped epitope. The results demonstrate the ease and precision with which the phage-displayed peptide sequences can be used to define and localize uncharacterized epitopes. The close similarity between the hexapeptides encoded by the four reactive phage is striking and suggests a high degree of specificity in the PAb240ligand interaction (Fig. 1). The presence of RHSV in all the phage sequences suggests an obligatory requirement, for this t,etrapeptide for ant,ibody binding. The C-terminal residue in the phage peptide sequences is either an isoleucine or a raline. Thus, although there is some flexibility at this posit tion, only closely related residues are Merated. (lonstraint a.t this terminal residue is consistent wit,h the lack of reaction with Xenopus ~53. which has a cysteine at, this posit’ion. Examination of all the peptidr sequences tested for P,4b240 reac%ivit,y shows ctonsiderable flexibilitj): in t.he first residue 01 the hexapeptide, wit,h positively and negatively charged residues, as well as aliphatic and aromatics residues being tolerated. However, in a,11four phagr sequences, the conserved arginine residue is in the second and not the first position in the peptide. An arginine in position one would be preceded by an alanine. present. in t.hr invariant flanking sequt~ncr. This may reflect a,n inability to tolrrat’e alanine in position one of the hexapeptide or. selection for peptides with highest affinities in which the arginine is preceeded by a large. hydrophobic residue (isoleucine, tryptophan or tyrosine). The cross-reactivity of monoclonal antibodies has been much discussed (f,ane & Koprowski. 1982: et trl.. 1982) a.nd it has been predicted that Crawford antibodirs recognizing simple, linear epit,opes will br cross-reactive simply due t,o the probahi&v of a given peptide sequence occurring more t,han once in the combined sequences of all existing probeins. Here we have shown t,hat such cross-react’ive prot,eins can indeed be identified on the basis of possession of’ the known rpitope sequence. f nterestingly, although t.he PAb240 epitope is cryptic: in correctly folded, wild-type 1~53. the results of the ELI&4 analysis suggest that thr epit*ope is exposed on thr surface of native TFIIIA. 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by A. R. Fersht