1Department of Virology, Haartman Institute, P.O. Box 21, FIN-00014 University of Helsinki, Finland and 3Microbiology and Tumour Biology Centre, Karolinska Institute and 4Swedish Institute for Infectious Disease Control, S-171 77, Stockholm, Sweden
2 To whom correspondence should be addressed. e-mail: tuomas.heiskanen{at}helsinki.fi
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Abstract |
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Keywords: epitope/hantavirus/peptide/phage/second-generation library
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Introduction |
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A second-generation phage library (SGL) with semi-random amino acids would be excellent for corroboration of the proposed alignments, because the aligned amino acids can remain unaltered or the change should be conservative. The whole family of peptides consisting of allowed amino acid substitutions, the supertope (Kramer et al., 1997), could then be defined. A further benefit of the SGLs is the possibility of improving the binding properties, mainly the affinity and the binding specificity, of the peptide or miniprotein displayed on the phage. The peptides selected from SGL might be recognized better by other MAbs and polyclonal antibodies having an overlapping binding site. More detailed analysis of the surface structure and the folding of the antigen is achieved if alignments to two different sites along the primary sequence of antigen could be shown. The specificity and the affinity of the peptides found originally, e.g. by pepspot analysis of coding sequences, might also be improved with SGLs.
Puumala virus (PUUV; genus Hantavirus, family Bunyaviridae) consists of the envelope glycoproteins G1 and G2, three negative-sense RNA segments encapsidated by nucleocapsid protein N and of the RNA-dependent RNA polymerase. The S (small) segment encodes N, M (medium) G1 and G2 and L (large) the polymerase (Schmaljohn et al., 1985; Plyusnin et al., 1996
). PUUV is the etiological agent of nephropathia epidemica, a mild European form of hemorrhagic fever with renal syndrome (HFRS) (Brummer-Korvenkontio et al., 1980
). Other pathogenic hantaviruses include European or Asian viruses, such as Hantaan virus, which causes HFRS (Lee and Lee, 1976
), and New World hantaviruses, e.g. Sin Nombre (SNV), which causes hantavirus pulmonary syndrome (HPS) (Nichol et al., 1993
). The ß3-integrins have been shown to mediate the entry of pathogenic hantaviruses (Mackow and Gavrilovskaya, 2001
).
The neutralization sites of PUUV and Hantaan viruses in glycoproteins G1 and G2 have been defined using MAb-escape mutants. The immunoprecipitation of PUUV and Hantaan virus glycoproteins by some neutralizing MAbs and MAb-escape mutant sites show that the conformational neutralization sites form a surface structure created by both glycoproteins (Arikawa et al., 1989; Antic et al., 1992
; Lundkvist and Niklasson, 1992
; Wang et al., 1993
). The proximity and the continuity of these surface structures are demonstrated by the pattern of inhibition of binding of PUUV-neutralization site-specific peptide inserts by all the existing PUUV-neutralizing MAbs (Heiskanen et al., 1997
). We have mapped some of these conformational neutralization sites of PUUV G1 and G2 with phage-displayed random peptide library and pepspot assays (Heiskanen et al., 1999
).
Now we have shown that by combining data from the reactivities of the SGL-derived peptides synthesized on membrane, it was possible to derive a peptide that had a significantly higher affinity to a neutralizing MAb 1C9 in the synthetic soluble form than the original parent peptide.
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Materials and methods |
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Monoclonal antibodies were produced and purified as described previously (Lundkvist and Niklasson, 1992; Lundkvist et al., 1993
). Peptides were synthesized as spots on a cellulose membrane (AIMS Scientific Products) using polyethylene glycol spacer with Abimed Autospot robot ASP 222 (Frank, 1992
). In order to form disulfide bridges, the peptides on the membrane were incubated overnight in deaerated 1 M ammonium bicarbonate solution. The soluble peptides were synthesized with Fmoc chemistry in a Model 433A peptide synthesizer (Applied Biosystems), the disulfide bridges were formed with hydrogen peroxide and the peptides were purified with reversed-phase chromatography. The quality and the cyclicity of the soluble peptides were assessed with matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry. The peptide ATCDKLFGYYER GIPLPCAL was also biotinylated on its N-terminus. The concentrations of the peptides were measured based on their calculated extinction coefficients. For the tryptophan-containing peptide FPCDRLSGYWERGIPSPCVR the extinction coefficient 5600 M1 was used (Creighton, 1984
) and for the peptide ATCDKLFGYYERGIPLPCAL the extinction coefficient was calculated by the ProtParam Tool program (ExPASy). PUUV grown in cell culture was partially purified by centrifuging through a sucrose cushion.
Construction of semi-random second-generation phage library
The semi-random peptide library was constructed on fuse 5 vector (Scott and Smith, 1990) essentially as described previously (Koivunen et al., 1994a
,b). The oligonucleotide 5'-CACTCGGCCGACGGGGCTTTTCCTTGTGATAGGCT TTCTGGGTATTGGGAGAGGGGTATTCCGTCGCCTTGTGTTCGGGGGGCCGCTGGGGCcgaa-3' including the underlined coding sequence for the peptide FPCDRLSG YWERGIPSPCVR selected from the X2CXC18X2 library (Heiskanen et al., 1999
) and primer 5'-TTCGGCCCCAG CGGCCCC-3' (Koivunen et al., 1994a
) were annealed, filled with polymerase to double-stranded form, restriction digested and ligated into the gene coding for pIII in fuse 5 vector. The coding sequence for the peptide FPCDRLSGYWERGI PSPCVR was synthesized with nucleotide mixtures containing 66% of the original nucleotide (A, C, G or T) and 34% of an equimolar mixture of A, C, G and T. In the third coding position, an equimolar mixture of G and T was used. The vector containing the library was transfected to MC1061 bacterial cells using 10 electroporations. The yield of the library was 2x107 clones. The library was amplified for
36 h in 400 ml of Luria broth.
Panning with extremely low amounts of MAb 1C9
The second-generation phage library was panned essentially as described (Heiskanen et al., 1999). In the first panning round, an aliquot of a library was pre-incubated with a PUUV-antibody-negative serum for 50 min and allowed to bind to MAb 1C9 for 40 min. The effect of the PUUV negative sera was controlled by panning without sera. In the absence of serum, the library bound 1 µg of MAb 1C9
1800 times the background and in the presence of serum
200 times. The percentage of serum in the liquid phase of the second panning reaction was 17% and in the third 35%. The reaction time of the amplified phage population with MAb 1C9 was also shortened in each panning round and the coating concentrations lowered. In the third round, the phages were allowed to react for 15 min with 40 pg of MAb 1C9. Clones were sequenced from the first panning without serum in the liquid phase of the reaction (MAb 1C9 8 ng/well), with serum (MAb 1C9 40 ng/well), from the second panning without serum (120 pg/well) and with serum (470 pg/well) and from the third with serum (40 pg/well) and without serum (40 pg/well), when the binding was at least about four times over the background.
Panning with two MAbs having overlapping epitopes
An aliquot of the second-generation library was allowed to react in the first panning round with MAb 4G2, in the second with MAb 1C9 and in the third again with MAb 4G2. In the first round the phages were pre-incubated with 5% PUUV-antibody-negative serum overnight at 4°C before binding to MAbs. In the second round, to the liquid phase of the panning reaction 10 µg/ml PUUV nucleocapsid specific bank vole MAb 5E1 was added; 17 000-fold more phages bound to 250 ng of MAb 1C9 than to 250 ng of MAb PUUV nucleocapsid protein specific bank vole MAb 1C12. In the third round, the phages were pre-incubated for 70 min with 10% PUUV-antibody-negative serum and allowed to bind 50 ng of MAbs for 70 min. MAb 4G2 bound 11 times more phages than the control MAb 1C12 and MAb 1C9 bound 190 times more than MAb 4G2. A few 4G2-selected phage clones were sequenced.
Binding of MAbs on peptides synthesized on membrane
The reactivity of peptides on spots was determined as described previously with some modifications. The membranes were blocked with 3% milk powder, 1% BSA in TBS for 1 h at room temperature or overnight at 4°C. The MAb 1C9 was diluted to 0.1 or 1 µg/ml in blocking buffer and incubated for 1 h. Detection with enhanced chemiluminescence was done as described (Heiskanen et al., 1999). X-ray films were exposed from 1 to 10 s so that the intensities of spots could be compared. For more quantitative comparisons the optical densities of spots were measured with NIH-image software.
Binding of phage clones to MAbs
The binding of phage clones to MAbs was determined as described (Heiskanen et al., 1997, 1999) with enzyme immunoassay (EIA) on 96-well plates (Nunc). Briefly, microtiter wells were coated for 1 h with 0.5 µg/ml MAb in PBS and blocked with 1% non-fat dry milk powder in PBST (0.05% Tween 20 in PBS) for 30 min. The phage clones were diluted in PBST and incubated for 4560 min over MAb-coated microtiter wells. The plates were washed between incubations at least six times with PBST (phosphate-buffered saline, 0.05% Tween 20). The phages bound on the MAbs on the wells were detected with horseradish peroxidase-conjugated anti-M13 antibody (Pharmacia Biotech) and with tetramethylbenzidine substrate. After stopping the reactions with 2 M sulfuric acid (100 µl), the absorbances were measured at 450 nm. The phages were titrated or their amount was estimated from a calibration curve based on EIA of phage-particles coated on a microwell plate.
Inhibition of binding of MAb 1C9 to PUUV particles
Microtiter wells were coated overnight at 4°C with partially purified PUUV (passage code: 638) diluted 1:150 in PBS (100 µl/well) and blocked by 1% NFDMP in PBST (200 µl/well) and washed with PBST. The different dilutions of MAb in PBST were mixed with an equal volume of a fixed peptide concentration in PBST and incubated for 30 min at 37°C. A 100 µl volume of each mixture was added to the wells (in triplicate) and incubated for 40 min at 37°C. The HRP-conjugated secondary antibody was added for 40 min. The assay was performed in a Biosafety Level-3 laboratory owing to handling of infectious virus particles.
Comparison of the binding affinities of the peptides by surface plasmon resonance
A BIAcore 2000 system was used for measurements. The MAb 1C9 was amine-coupled to a CM5 sensor chip (Biacore) according to the manufacturers instructions at a concentration of 50 µg/ml in 10 mM acetate buffer, pH 5.2. The coupling level was 1400 resonance units (RU) after blocking of the active coupling surface with ethanolamine. The empty activated and blocked channel was used as the background surface for binding constant determination. HBS buffer (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20; Biacore) was used as running buffer both in coupling and in the determination of binding constants of the soluble peptides to MAb 1C9. The flow rate was 10 µl/min. The injection volume was 50 µl and the dissociation time 600 s. The bound peptides dissociated nearly completely during the dissociation time, so further washes with special, e.g. low-pH, buffers between injections were not needed. The background was subtracted before calculation of kinetic constants using Biaevaluation 3.0 software and the Langmuir 1:1 binding model.
The binding of soluble MAbs to the N-terminally biotinylated modified peptide immobilized on a sensor chip was measured as follows: the biotinylated modified peptide ATCDKLFGYYERGIPLPCAL was injected at a flow rate of 5 µl/min into a streptavidin sensor chip (Sensor Chip SA; Biacore) in HBS buffer, which resulted in a binding level of 400560 RU. A flow channel with only streptavidin was used to measure background binding. Then 140 nM MAb 1C9 in HBS buffer was injected into the biosensor at a flow rate of 5 µl/min. The sensor chip was regenerated with 1 M NaCl, 50 mM NaOH between injections. Furthermore, 667 nM MAb 4G2 was injected into the sensor chip at a flow rate of 5 µl/min in 20 mM TrisHCl, pH 8.0, 150 mM NaCl. The modified peptide on the sensor chip was reduced with a 5 s injection of 500 mM dithiothreitol (DTT) and subsequently 667 nM MAb 4G2, 667 nM control MAb 1C12 and 70 nM MAb 1C9 were injected in 5 mM DTT, 20 mM TrisHCl pH 8.0, 150 mM NaCl buffer.
Comparison of the second-generation phage library derived sequences with PUUV G1 and G2 glycoproteins
The peptides were compared with the amino acid sequence of the M segment of PUUV Sotkamo strain with the LALIGN program, which finds the best local alignments between two sequences (www.ch.embnet.org/software//LALIGN-form.html).
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Results |
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The proportion of the equimolar nucleotide mixture (34%) compared with constant nucleotide originally present in the parent phage-clone nucleotide sequence (66%) produced a 20-mer peptide library in which the amino acid sequence of the original clone would be represented at an estimated ratio 1:150 000. The semi-random second-generation library that we generated had two properties. The probability of an amino acid change depended on the amino acid the original codon was coding for. Also, a change to an amino acid in which two nucleotide changes are needed was less probable than a change where only one change is needed. The same rules are valid in nature provided that every mutation is as probable as any other.
Panning with extremely low target molecule amounts
In order to isolate a higher affinity peptide, we coated the microtiter wells with lower and lower amounts of the neutralizing G2-specific MAb 1C9 in later panning rounds. Binding to picogram quantities of MAb 1C9 was achieved. Addition of PUUV-antibody-negative human serum to the liquid phase of the binding reaction was intended to increase the specificity of the selected insert and shortening of the binding time on later panning rounds corresponded to the selection of peptide inserts with a faster association rate to the ligand, MAb 1C9. MAb 1C9 was panned in the absence of serum in the liquid phase of microtiter wells for comparison.
Sequences selected with MAb 1C9
The SGL defined the amino acids absolutely needed for binding of MAb 1C9 in the phage environment. The variability of an amino acid in the phage environment (Table I) should be inversely correlated with the intensity of the MAb 1C9 reactivity in the alanine scan of the peptide cc5 (Figure 1). Some directionality in changes was seen, which may be due to improved binding of phages in selection conditions. In the conserved motif the change of R-5 to K was accompanied by the appearance of F in position 6 or 7 in pannings with picogram amounts of MAb 1C9 in the presence of serum. The L-6 changed twice to F. In the presence of serum in the selection with 40 pg of MAb 1C9, the residue F-1 was changed to Y in the sequenced inserts. The change of serine-16 to leucine between important prolines was favored at higher coating concentrations.
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In some binding experiments we had noted that the other neutralizing MAb 4G2, having an overlapping epitope with MAb 1C9 in G2 glycoprotein, also seemed to bind the phage-displayed peptide insert FPCDRLSGYWERGIPSPCVR when the amount of phages was increased sufficiently. To improve this property we panned the secondary phage library first against MAb 4G2, then against MAb 1C9 and again against MAb 4G2. In the third panning round, phages bound 50 ng of MAb 4G2 11 times and MAb 1C9 2000 times more than the PUUV nucleocapsid protein-specific MAb 1C12.
Sequences selected with MAbs 1C9 and 4G2
Surprisingly, the sequences of the MAb 4G2-selected phage clones did not show any new selected amino acid in common (Table II). The binding motif between the cysteines remained essentially unchanged in the four sequenced clones. The P-2 was absent from the sequenced inserts and thus selected against and was replaced twice by serine or threonine. The sequences selected by MAb 4G2 showed higher conservation of the original motif than the selections with a low concentration of MAb 1C9.
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The binding-determining motif of the peptide FTCDRLS GYWERGIPLPCGL (cc5) selected with MAb 4G2 (Table II) was studied with peptides synthesized on membrane (Figure 1). The variability of an amino acid in the phage environment (Tables I and II) should be inversely correlated with the intensity of the MAb 1C9 reactivity (Figure 1). The parent peptide (P) gave as strong reactivity as the peptide cc5. For the motif GY-ERGIP-P any change to alanine abrogated the binding to MAb 1C9. The change of D-4 almost completely removed the binding, while the changes C-3 and L-5 removed at least half of the binding. The F-1 change to alanine increased the binding of the insert cc5 to MAb 1C9. Also, the L-16 change to A did the same although not as clearly. Interestingly, the change of the last cysteine to alanine did not have any effect.
Epitope minimization
The minimal region of the peptide FTCDRLSGYWER GIPLPCGL (cc5) needed for binding to MAb 1C9 was determined by deleting amino acids one by one starting from both the N- and C-terminal ends (Figure 2). Removal of the CGL sequence from the C-terminal side had no detectable effect. Removal of P-17 abolished binding. Removal of C-3 from the N-terminal side decreased and removal of Y-9 eliminated the reactivity altogether. This contrasts with the alascan where the disappearance of D-3 had a much larger effect. According to this deletion series the C-terminal motif YWERGIPLP is an independent binding unit.
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We compared the binding affinities of the SGL-derived phage clones with MAb 1C9 coated on microtiter wells (50 ng/well). The clones 10, 13 and 14 selected with 40 pg of MAb 1C9 (Table I) bound MAb 1C9 with 108 transforming units (TU) and the parent peptide with
109 TU. Hence, the rather diverse sequences from the last panning round with MAb 1C9 (40 pg) did not show any clear differences between each other in binding as phage clones although they bound with apparently higher affinity to MAb 1C9 than the parent phage clone (data not shown).
The peptides selected with 40 pg of MAb 1C9, the parent peptide and two of the peptides selected with 4G2, cc5 and cc6, were synthesized on the membrane (Figure 3). The concentration of the detecting MAb was lowered to 0.1 µg/ml in order to make the differences in binding affinity more visible. The peptide 14, YPCDRLAGYYERGIPSPCVR, bound several times better and the peptide 10, LPCDKLFGYWER GIPYPCVL, slightly better than the parent peptide. The peptide 14 had three differences compared with the parent peptide, Y-1, A-7 and Y-10. The first one is not significant because other peptides with this change did not show any improved binding. The A-7 change is not significant because derivatives of peptide 14 without A-7 and with Y-10 still showed the same level of binding improvement (data not shown). Thus only the Y-10 was a significant change.
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Based on the binding reactivities of the peptides synthesized on the membrane, the frequencies of the selected changes in the second-generation phage population and similarities of the selected changes in PUUV G1 and G2 proteins, the peptide ATCDKLFGYYERGIPLPCAL was designed. This peptide blocked the binding of MAb 1C9 to PUUV particles more effectively than the parent peptide (Figure 4). The binding constants of both of the peptides were determined with surface plasmon resonance. The modified peptide associated more rapidly with MAb 1C9 coupled to the sensor chip and dissociated more slowly (Table III).
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Significance of the disulfide bridge
In order to study the importance of the possible disulfide bridge on pepspot reactivities of SGL-derived and also of modified peptides, the cysteines in the modified peptide were changed to serines, structurally similar amino acids. When detected with 0.02 µg/ml MAb 1C9 the peptides ATCDKLFGYY ERGIPLPSAL and ATSDKLFGYYERGIPLPSAL synthesized on the membrane reacted as well as the modified peptide ATCDKLFGYYERGIPLPCAL (Figure 5). This shows that for the reactivity of the peptide on the membrane the cysteine bridge is not significant but the SH or OH group in position 3 is, since a change to alanine decreased the reactivity by half (Figure 1). The association rate of the MAb 1C9 with the reduced and biotinylated ATCDKLFGYYERGIPLPCAL on the streptavidin sensor chip on the biosensor seemed to be at the same level as association with the cyclic ATCDKLFGYYERGIPLPCAL, whereas dissociation was much more rapid (data not shown). The contrasting lack of reactivity difference of C S mutants further indicates that the disulfide bridge was not formed on the membrane.
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The MAb 4G2 selected clone FTCDRLSGYWERGIPLPCGL (cc5) bound MAb 1C9 10 times more and MAb 4G2
104 times more than the parent clone (Figure 6). This showed that the selection had had an effect on the binding properties and that the terminal amino acids outside cysteines (FT- and -GL in cc5) are important for presentation of the essential binding motif, probably by lowering the proportion of the cyclic peptide in the phage population compared with the proportion of the linear. MAb 4G2 did not react with the cyclic parent peptide (P) coated on a microtiter well (data not shown) or with the N-terminally biotinylated cyclic parent peptide on a streptavidin-coated plate, as shown previously (Heiskanen et al., 1999
). The same applied to the modified peptide ATCDKLFGYYERGIPLPCAL. However, when the N-terminally biotinylated modified peptide was coupled to the streptavidin-sensor chip the MAb 4G2 reacted clearly with both the cyclic and the reduced modified peptide whereas the same concentration of control bank vole MAb 1C12 did not bind the reduced modified peptide. The binding and the dissociation of the reduced and the oxidized peptide were at the same level (data not shown).
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Hypothetically, if a change occurs in the binding-determining motif and the SGL sequence still binds to the MAb used for selection, changes outside the motif may have had a compensatory effect. As a test for this, the amino acids E-1 and I-2 in sequence 1 (Table I) were introduced to positions 1 and 2 of the modified peptide, thus forming the peptide EICDKLFGYYERGIPLPCAL. This gave a higher signal in pepspot assay than the modified peptide (Figure 5), indicating that the EI dipeptide compensated the changes R-5 to P-5 and E-11 to W-11 in peptide 1 (Table I). In contrast, the change DRLS DRMS in the motif diminished binding (Figure 5).
Sequence alignments with PUUV glycoproteins
The GY in the parent peptide sequence is necessary for the second-generation library peptides to bind MAb 1C9, which is consistent with the previously shown alignment to the site 931934 in PUUV glycoprotein G2 (Heiskanen et al., 1999). The N-terminus ATCDKLF of the modified peptide ATCDKLFGYYERGIPLPCAL is similar to the sequence ADSDKIF (10817) in the C-terminal region of G2. However, when T-2 is replaced by D in the modified peptide, the binding is diminished. The C-terminal part of the peptide is similar to the sequence WTGFIPLP (3707) present in G1. Interestingly, the F to A change in the N-terminus of cc5 increases binding (Figure 1). The ATC sequence is similar to the N-terminus of the peptide ASCPVQQYPSGPCEASVCDT selected with MAb 4G2 from the phage-displayed random peptide library. This peptide reacts with MAbs 1C9 and 4G2 equally well in pepspot assay and it can be aligned with the linear PUUV G2-peptide FATTPVCQFDGN (residues 917928, similarity underlined) reacting with MAbs 4G2 and 1C9 (Heiskanen et al., 1999
).
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Discussion |
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The parent peptide FPCDRLSGYWERGIPSPCVR was selected as the basis for the SGL because it had the highest affinity when synthesized on the membrane of all the sequences selected by PUUV-glycoprotein-specific neutralizing MAbs.
The SGL selection with MAb 1C9 defined the changes, which do not abolish the binding to MAb 1C9 in the sequence family derived from FPCDRLSGYWERGIPSPCVR and thus the allowed variability or supertope (Kramer et al., 1997) for this peptide as expressed on the N-terminus of the pIII protein of fd-tet. The peptide on the phage and the peptide on the cellulose membrane did not have identical properties. The variability of an amino acid position in the SGL (Tables I and II) should correspond to the intensity of a spot in the alanine scan in Figure 1. In contrast, the amino acids R-5, W-10 and C-18 were not changed in the library or were changed less than expected by chance, but they could be changed to alanine in pepspot without any apparent loss of reactivity. Tryptophan is more common in phage-displayed inserts than expected. This might be due to its unspecific binding force, affinity to hydrophobic surfaces and clefts on the proteins and other surfaces. The appearance of Y in to the first position of the peptide inserts in the presence of serum selection may reflect also these factors or it may have an effect on disulfide-bridge formation.
The binding-affinity contributing factors for a peptide expressed on phages are cyclicity, multivalency and the possible conformation-stabilizing and -forming effect of the phage environment (Kischenko et al., 1994; Jelinek et al., 1997
). The peptides synthesized on spots are multivalent (the density is sufficient for binding the bivalent antibody on both sites) but the disulfide bridge did not seem to be formed in peptides cc5 (FTCDRLSGYWERGIPSPCGL) and ATCDK LFGYYERGIPLPCAL synthesized on the membrane. This lack of one binding-affinity-increasing factor increases the stringency towards individual amino acids so that replacements are less tolerated than in the cyclic peptide, which is shown by the smaller essential binding motif defined by SGL compared with the motif defined by alanine scanning. For example, E-11 belonging to the necessary binding motif according to the alascan was changed to G in sequence 7 (Table I). The biosensor experiments with the cyclic and linear forms of the peptide ATCDKLFGYYERGIPLPCAL indicate that the binding-enhancing effect of the W-10 to Y change is at least partially masked by the virtually non-existent dissociation of the phage-displayed peptides from MAb 1C9 having the supertope defined by SGL selection and that the changes increasing the affinity of linear peptide increased also the affinity of the corresponding cyclic peptide. Cyclization is known to increase the binding affinity of, e.g., the integrin-binding RGD peptides (Koivunen et al., 1993
). Antibodies have a flexible hinge and many antibodies undergo a conformational switch when they bind to, e.g., viral antigen (Smith et al., 1996
). It may be that a cyclic peptide is able to induce this binding switch due to its rigidity and a linear and flexible peptide is not. It seems that in later panning rounds changes improving binding to MAb 1C9 accumulated in the sequences, but that these sequences did not become dominant, because the dissociation of all the sequences was very low and part of the changes simply improved the retention of phages in the microtiter well without increasing the affinity to MAb 1C9. The selection with MAb 1C9 thus achieved the upper limit of affinity with this phage display system and an analysis of binding in an independent environment, peptides synthesized on membrane, was needed to detect the affinity to MAb 1C9 increasing changes.
The selection with MAb 4G2 showed that the MAb 1C9-recognizing sequence FPCDRLSGYWERGIPSPCVR selected independently of MAb 4G2 already resembled the surface structure of the common binding site of these MAbs in PUUV G2, because the binding motif was conserved (Table II) and this motif was recognized only by MAb 4G2 having an overlapping epitope with MAb 1C9 and not by others. The selection with MAb 4G2 affected the way of presentation of the the basic motif by changing the first two amino acids before the first cysteine. These two first amino acids determine the propensity for disulfide-bridge formation. On the phage surface there probably exist both disulfide-bridged and linear forms of the peptides (Bonnycastle et al., 1996) and MAb 4G2 selection favors the linear form. The increased reactivity of MAb 4G2 selected sequences towards MAb 4G2 as compared with MAb 1C9 would then be due to a higher proportion of linear peptide on the surface of the phage compared with cyclic peptide.
In conclusion, semi-random second-generation phage libraries are useful tools for improvement of affinity and fit of the found binding peptides for target molecules and for definition of the supertope of the binder. Paradoxically, such structural constraints as disulfide bridges seem to counteract the selection of new affinity-increasing amino acids by lowering the dissociation rate, which should be taken in account in library planning and selection of sequences. Our results show that the affinity-increasing changes in the linear form of the peptide can increase the affinity compared with the original cyclic peptide when the peptide is again cyclized. Also, the study of binding of the selected peptide inserts in a system unrelated to the phage environment, such as the pepspot assay, may greatly facilitate the improvement of the binding affinity of the peptide inserts.
The advantage of the phage display is the vast diversity of the expressed libraries, which results in the easiness of finding several different peptides binding to the same site, e.g. in a neutralizing antibody. Some of these peptides representing different supertopes with different binding motifs are likely to resemble more closely real interacting molecules, e.g. virus surface, than others. Our approach, the use of phage display of random peptides, the generation of SGLs based on found peptides and the systematic amino acid replacements in peptides synthesized as spots on a membrane, is generally applicable to increase the affinity and to change and to study the binding specificity of ligand-binding peptides. From found binding partners one can select those which either resemble or do not resemble structurally the natural interacting molecule and they can be modified in either direction according to need. Practical applications would include the identification of leads for vaccine and drug development.
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Acknowledgements |
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Received October 18, 2002; revised April 16, 2003; accepted May 26, 2003.