Selection of peptides inhibiting a b-lactamase-like activityAnne-Sophie Yribarren, Daniel Thomas, Alain Friboulet and Be´range`re Avalle Ge´nie enzymatique et cellulaire, UMR 6022 CNRS,
Trang 1Selection of peptides inhibiting a b-lactamase-like activity
Anne-Sophie Yribarren, Daniel Thomas, Alain Friboulet and Be´range`re Avalle
Ge´nie enzymatique et cellulaire, UMR 6022 CNRS, Universite´ de Technologie de Compie`gne, France
A library of random peptide sequences was used to select
peptides that inhibit an anti-idiotypic catalytic Ig,
immuno-globulin (IgG) 9G4H9, with a b-lactamase-like activity This
library displays cyclic heptapeptides on the surface of
bac-teriophages and represents a collection of up to 4.5· 109
peptides The first selection step aimed at enriching the
lib-rary in species that bind to the whole Ig molecule The
sec-ond step was to discriminate peptides that bind to part of the
molecule other than the active site Selected peptides were then screened by surface plasmon resonance analysis Those displaying measurable Kd values were assayed for their ability to inhibit the catalytic Ig
Keywords: catalytic antibodies; anti-idiotypy; b-lactamase; phage display library; constrained peptide
The elucidation of the rules governing the relationships
between the structure of proteins and their function is
currently one of the major challenges In this field,
combinatorial approaches offer tremendous possibilities
The main requirement is to be able to sort, from the
engineered diversity, mutants or new compounds according
to the required function
Random peptide libraries are inexhaustible sources of
small compounds likely to bind targets such as enzymes,
antibodies, receptors or even DNA (recently reviewed in [1])
Combinatorial libraries can be chemically synthesized or
displayed on engineered bacteriophages, as proposed by
Smith’s pioneer studies from the 1980s [2,3] The use of
phages allows one to workon huge libraries It allows rapid
isolation of the sequence of the selected compounds, leading
to the identification of residues involved in the interaction [4]
We have developed previously an original approach to
generate catalytic proteins, other than enzymes, yielding
valuable information about structure–function relationships
[5–8] According to Jerne’s concept [9], some of the
anti-idiotypic Igs generated by immunization by antibodies may
mimic some of the antigenic properties and functions These
antibodies are said to be the internal images of the original
antigens We applied this theory to the elicitation of a new
catalyst, displaying a b-lactamase-like activity Starting
from b-lactamase, we have produced an inhibitory active
site-specific Ig (Ab1), capable of inducing anti-idiotypic Igs
(Ab2) displaying the model-like catalytic activity [10] We
have also demonstrated that the b-lactamase-like Ig (IgG
9G4H9) is a true anti-idiotypic Ig by generating polyclonal
Ab3, which recognizes b-lactamase [7] The Ab2 catalytic Ig
would then be of valuable help to elucidate on the one hand
some structure–function relationships associated with b-lactamase activity and, on the other hand, the mecha-nisms governing the transfer of catalytic information throughout the idiotypic network
In this context, we proposed to achieve the selection of peptide inhibitors, specific for the activity observed in the b-lactamase Ig We have carried out a two-step selection procedure from a random library of constrained peptides displayed on bacteriophages, based on exclusive affinity for the Ig active site
Materials and methods Production and purification of IgG 9G4H9 Hybridomas were grown in Dulbecco’s Modified Eagle’s Medium (Eurobio) containing 10% (v/v) fetal bovine serum, 1% (w/v)L-glutamine 200 mM, 1% (w/v) pyruvate
100 mM, 50 UÆmL)1 penicillin and 50 lgÆmL)1 strepto-mycin Harvested cells were expanded by injection into pristane (2,6,10,14 tetramethyl pentadecan)-treated BALB/
C mice Resulting ascitic fluids were collected 10 days later, centrifuged at 350 g for 5 min at 4C and stored at)20 C Purification of monoclonal Igs was carried out on a HiTrap rProtein A column (Amersham Pharmacia Biotech) that presents a high affinity for the constant region of IgGs
of isotype 2b, such as 9G4H9 The binding buffer contained
50 mMTris/HCl, pH 9, and 0.2MNaCl The elution buffer was obtained from Biorad and was used according to the manufacturer’s instructions The IgG-containing fractions were dialysed overnight against a 0.1Msodium phosphate buffer, pH 7.4 After concentration by centrifugation at
7000 g for 45 min on 30 kDa MicrosepTM filters, IgG concentration was determined by the bicinchoninic acid assay (BCA, Sigma) The purity of the preparation was estimated by silver-stained 8% (w/v) polyacrylamide gel electrophoresis
Amplification and purification of phage peptide libraries The random cyclic heptapeptides library (named CX7C) was supplied by E Koivunen, Dept of Biosciences, Division
Correspondence to B Avalle, Ge´nie enzymatique et cellulaire,
UMR 6022 CNRS, Universite´ de Technologie de Compie`gne,
BP 20529, 60205 Compie`gne Cedex, France.
Fax: + 33 3 44 20 39 10, Tel.: + 33 3 44 23 44 12,
E-mail: avalle@utc.fr
Abbreviations: TU, transforming units; SPR, surface plasmon
resonance; BLIP, b-lactamase inhibitory protein.
(Received 6 March 2003, revised 15 April 2003, accepted 6 May 2003)
Trang 2of Biochemistry, University of Helsinki, Finland [11] The
library was amplified by infection of mid-log phase
Escherichia colicells by 3.1010of phage transforming units
(TU) for 30 min, without shaking, at 37C The infected
bacteria were then grown overnight, shaken at 37C in 1 L
Luria-Bertani medium containing 20 lgÆmL)1tetracycline
The amplified phages present in the culture supernatant
were precipitated overnight at 4C by adding 20% (v/v) of
20% polyethylene glycol 8000, 2.5M NaCl (PEG/NaCl)
Phages were then pelleted by centrifugation at 10 000 g for
45 min at 4C, suspended in 10 mL deionized water and
filtered through a 0.45 lm filter unit (Millipore) The phages
were reprecipitated in PEG/NaCl for 1 h at 4C The
library was finally recovered in 1 mL Tris-buffered saline
(TBS), pH 7.5, containing 50 mM Tris/HCl and 150 mM
NaCl The library was stored at)20 C
Titration of the library was performed by infection of
mid-log phage E coli bacteria by dilutions of phages for
30 min at 37C without shaking One hundred microlitres
were then plated on solid Luria–Bertani agar plates
containing 20 lgÆmL)1tetracycline The plates were
incu-bated overnight at 37C and visible clones counted
Solid phase selection procedures
Experimental concentration conditions are summarized in
Table 1
Positive selection Maxisorb plates (96 wells) were
pur-chased from Nunc The panning method was derived from
Parmley and Smith [3] Unless otherwise stated, each step of
the procedure was followed by three washings of the plates
in TBS containing 0.1% Tween 20 (TTBS) and plates were
incubated 1 h at 37C without shaking
The microplates were coated with 100 lL of 2.5 lgÆmL)1
rat anti-(mouse IgG) in 0.1M carbonate buffer pH 8.6
Wells were then saturated with 100 lL of 2% milkin NaCl/
Tris (MTBS) (w/v), and 100 lL of 1–10 lgÆmL)1 IgG
9G4H9 in NaCl/Tris were added to the saturated wells
Between 5· 1010 and 2.5· 1011TU were first incubated
for 10 min at 37C in MTBS, to avoid the binding of
displayed peptides to milkproteins This solution was then
transferred to the plate and incubated for 2 h at 25C This
step was followed by 10 TTBS washings, each washing lasting at least 5 min The bound bacteriophages were eluted from the plates by a 10 min incubation at room temperature with a solution containing 0.1MHCl adjusted
at pH 2.2 by the addition of solid glycine The pH of the collected phage solution was neutralized by 11% of 2MTris (v/v)
The enrichment of the starting library in phage specific for the IgG was followed by titration of the solutions before and after each round of selection This is expressed as the following ratio: TU at round (n)/TU at round (n)1) After each round of selection, eluted phage were ampli-fied by infection of 20 mL mid-log phase bacteria as described below, then used for the subsequent round
Negative selection This selection procedure was derived from the positive one The steps and washings were the same, but the IgG 9G4H9 was exposed to contact with penicillin G under conditions described previously [12] The covalent complex thus formed was added to saturated wells The bacteriophage issued from the positive selection that were not retained on this complex, i.e that are not specific for the active site, were directly recovered and amplified for further rounds of selection
Phage ELISA Unless otherwise stated, wells were filled with 100 lL of the indicated solutions, plates incubated for 1 h at 37C and each step followed by three washings in TTBS
One microgram IgG 9G4H9 in 0.1Mcarbonate buffer,
pH 8.6, was coated on a Maxisorb microplate Wells were saturated by MTBS Variable amounts of bacteriophage, from 5· 107to 5· 1010TU per well, were then added and incubated for 2 h in MTBS at room temperature After 10 washes in TTBS, bound phage were detected by 1 lg M13 horseradish peroxidase-conjugated monoclonal anti-bodies (mAbs) (Amersham Pharmacia Biotech) The fixation of labelled antibodies was measured by the addition of 0.3 mgÆmL)1azino ethylbenzthiazoline sulfonic acid (Sigma), a substrate of peroxidase, in 50 mM citrate buffer, pH 5, with 0.05% H2O2 Absorbance was read at
405 nm
Sequencing of phage DNA Oligonucleotides were purchased from Oligo Express and DNA sequencing was performed by Genome Express (both Paris, France)
The DNA peptide inserts of individual plated clones issued from rounds of selection were amplified by PCR performed on an Eppendorf thermocycler A precycle at
94C for 10 min and 35 cycles of denaturation at 94 C for
1 min, AB 348 and AB 349 primers annealing at 54C for
1 min and elongation at 72C for 1.5 min Taq DNA polymerase was purchased from New England Biolabs The forward primer AB 348, 5¢-TTAGCAAAACCTC ATACAGAA-3¢, and the backward primer AB 349, 5¢-GATGCTGTCTTTCGCTGCTGAG-3¢, were used for
5¢-ATTCACCTCGAAAGCAAGCTG-3¢, was used for sequencing
Table 1 Experimental conditions of selection Concentrations of rat
anti-(mouse IgG), IgG 9G4H9 and phages from the library CX 7 C are
indicated from the first to the sixth round of positive selection on IgG
9G4H9 and for the two rounds of negative selection on IgG 9G4H9
complexed to its suicide substrate penicillin G The terms in brackets
indicate the name of the round used in the text.
Catalytic IgG 9G4H9 (lgÆmL)1)
CX7C library ( M )
Trang 3Synthetic peptides
Several selected peptides displayed on the surface of
bacteriophages were synthesized in their soluble form The
cyclic and biotinylated peptides containing 18 amino acids
were purchased from Eurogentec (Angers, France) and
designed with the two N-terminal residues belonging to the
pIII protein of the bacteriophages These were followed by
two cysteines flanking the peptide sequence The C-terminal
sequence corresponds to the biotin moiety (linked to the
lysine of Gly-Ala-Ala-Gly-Ala-Glu-Lys) The sequence of
free peptides is thus: NH2GACX1X2X3X4X5X6X7CGAA
GAEKCONH2, with Xn corresponding to the random
residues of the displayed peptide Synthesis was performed
under oxidative conditions, to preferentially form the
cyclizing disulfide bond Between 1 and 3 mM of peptide
stocksolutions were stored at)20 C and prepared in water
or in 50 mM acetic acid solution according to their
hydrophobic features
Surface plasmon resonance (SPR) analysis
SPR analysis is performed with a BIAcore X (Biacore AB,
Sweden) Streptavidin coated chips and running buffer HBS
pH 7.4 containing 10 mM Hepes, 0.15M NaCl, 3.4 mM
EDTA and 0.005% surfactant P20, were from Pharmacia
(Uppsala, Sweden)
Immobilization of the biotinylated ligands (40 lgÆmL)1
in HBS) on the surface of SA chips was done by injecting
40 lL at a flow rate of 5 lLÆmin)1 The Kd values were
determined by injecting 40 lL of Ig solutions ranging from
100 to 600 nMin HBS at a flow rate of 7 lLÆmin)1 The
regeneration of the surface was carried out by injecting
6 lL of 50 mM HCl Curve fitting and kinetic data
analyses were carried out using BIAEVALUTION 3.0.1
software
Kinetic assays
The catalytic activity of 2 lMIgG 9G4H9 was measured
spectrophotometrically by following the hydrolysis of
ampicillin at 232 nm in 0.1M phosphate buffer (pH 7.4)
IC50 values were determined on the basis of the residual
activity of 9G4H9 after a 20 min incubation with different
amounts of peptides or phage
Results
Selection of peptides binding to the Ig
The library (CX7C) of constrained heptapeptides displayed
on the surface of bacteriophages was kindly provided by
E Koivunen [11] Its diversity extends to 4.5· 109different
combinations During the amplification and propagation of
phage, we always ensured that the whole diversity was
expressed by titrating the purified phage
The first step of the selection procedure was performed on
plates coated with monoclonal antibodies directed against
the constant region of mouse IgGs, as 9G4H9 The active
site within the variable region of 9G4H9 should thus be
favorably exposed to the library We also confirmed that
9G4H9 activity was fully maintained in TBS
From the third round, the selection pressure was increased by reducing the concentration of 9G4H9 from
10 to 1 lg (Table 1) The peptides selected from this third round should therefore display improved affinities for the
Ig After five rounds, the yield of specific peptides retained
on 9G4H9 was 1000-fold higher than in the original library
As shown in Fig 1 (abscissa R 1 to R 5), a significant enrichment of specific peptides was obtained from the second round The signals measured in phage ELISA from the first to the sixth round (Fig 2, abscissa R 1 to R 6) indicate that the affinity of the pool of selected peptides progressively increases
We randomly chose 27 isolated clones from the fifth round, which were amplified and sequenced These peptides could then be gathered in 10 families according to their amino acid content (Table 2) Within each family, the peptides share the same sequence The analysis of the
Fig 1 Enrichment of the CX 7 C library in phages recognizing the target The yield of recovered TU corresponds to the ratio of phages collected
at the end of each round over phages inserted in this round Open bar: Phages recovered during the positive steps of the selection (rounds R1
to R5) carried out on IgG 9G4H9 (Grey bar): Phages recovered after the negative selection (rounds NS 1 and NS 2), i.e with IgG 9G4H9-penicillin G covalent complex as a target.
Fig 2 Phage ELISA on enriched CX 7 C library at each round of selection Values of absorbance at 405 nm correspond to the fixation of
5 · 10 10
M TU to 1 lg of immobilized IgG 9G4H9, revealed by anti-M13 horseradish peroxidase-conjugated mAbs in the presence of azino ethylbenzthiazoline sulfonic acid Open bar represents the phages of the positive step of selection (R 1 to R 6) (grey bar) those of the negative step (NS 1 and NS 2), and blackbar the original library The background signal corresponds to the fixation of CX 7 C to the microplate not coated with IgG 9G4H9 Standard deviations are cal-culated from three independent experiments.
Trang 4aligned sequences, notably the frequency of amino acids at
each position, provided two pieces of information First, the
three central residues X3X4X5 (notation as in Synthetic
peptides section) are mostly hydrophobic (more than 59%
of the considered residues) Trp is present in 19% of these
three residues, Leu in 11% and Met and Phe in 7% Second,
the Pro is present in 18.5% of the peptide sequences
Binding of the peptides displayed on phages to IgG
9G4H9 was measured by a monoclonal phage ELISA
Clones from the fifth round of positive selection were
assayed in addition to the original library CX7C (Fig 3)
They all showed a significant capability to bind the Ig The
library gave a weaker ELISA signal than isolated clones that
have encountered five rounds of selection because it
contains many phage that are not specific for the Ig This
first positive step of selection is consequently efficient,
because it allows one to obtain peptides that significantly
bind to the target
Selection of peptides binding to the Ig active site
The second step of the procedure, called negative selection,
was carried out on phage from the fifth round of positive
selection This step was applied using IgG 9G4H9 whose
catalytic site was covalently blocked by penicillin G Previous studies on 9G4H9 mechanism have shown that hydrolysis of penicillin G by the Ig behaves as a suicide substrate mechanism by rapidly forming a covalent irre-versible complex [12] This property was thus exploited to select peptides with inhibitory features Peptides that both (a) bind to the Ig and (b) do not bind to the Ig-suicide substrate complex should be specific for the Ig active site They were then amplified prior to another round of negative selection Figure 1 (abscissa NS 1 and NS 2) shows that the two rounds of negative selection were efficient in enriching the yield of peptides not retained to the Ig inactivated by penicillin G, i.e specific for the active site The phage ELISA carried out on the free IgG 9G4H9 (Fig 2, abscissa
NS 1 and NS 2) gave significant signals, meaning that some
of the peptides isolated from the two negative rounds remain specific for the Ig The results were compared with those of the sixth round of positive selection (R 6): no gain
of affinity for this round of selection was observed whereas the affinity was improved for the two rounds of negative selection However, no significant difference could be observed between the first and the second round of negative selection in our experimental conditions On the one hand, there is an increase in the yield of specific peptides recovered, but on the other hand the affinity remains the same Nevertheless, increasing the selection pressure by decreasing the amount of Ig linked to its substrate should not be efficient in improving affinities because, for negative selec-tion, only peptides that are not retained are collected
We isolated and sequenced clones from the two rounds of negative selection Peptides issued from the negative selec-tion display a high percentage of Trp residues (13%) (Table 3) Three families of peptides previously sequenced
at the fifth round of positive selection were retrieved in the first round of negative selection The three families of peptides have a Pro at their C-terminal (X6or X7), and they contain a Trp More than 38% of the residues are hydrophobic Two families (2 and 5) were also retrieved in the second round The peptides of these two families
Table 2 Amino acid sequences of the 10 families of the 27 selected
clones Clones isolated from the five first rounds of positive selection
were isolated and sequenced.
Fig 3 Phage ELISA on clones isolated from the fifth round of positive
selection The name of the clone (F x ) corresponds to the family
reported on Table 2 to which belongs the clone Open bar corresponds
to the signal given by isolated clones and blackbar corresponds to the
signal obtained with the library CX 7 C Absorbance values at 405 nm
are the means of three identical experiments carried out independently.
Error bars indicate standard deviations.
Table 3 Sequences of clones issued from the negative selection Family
(positive selection) First round Second round
Name of the free corresponding peptide
GWVGQLG SAKYALW RSQGTLE PLDLLPL
a
Sequences that were already identified after the fifth round of positive selection b Sequences that are retrieved in both rounds of negative selection.
Trang 5appeared clearly as good candidates for the inhibition of the
catalytic activity shown by IgG 9G4H9
Analysis of binding properties by surface plasmon
resonance
Phage ELISA appeared to be an efficient qualitative assay
However, it cannot be used to measure accurate Kdvalues
because the affinity of the Ig for phage particles probably
takes part of the signal We thus worked on synthetic free
cyclic peptides
The free structures corresponding to the sequences of
seven negatively selected peptides (Table 3), displaying the
best signals in phage ELISA, were synthesized according to
the following feature: NH2GACXnCGAAGAEKCONH2,
with Xn corresponding to the residues of the displayed
peptide The synthesis was performed under oxidative
conditions in order to induce the formation of the disulfide
bond by association of the two bordering cysteines, thus
leading to a cyclic structure This cyclic structure is flanked
at the N-terminal end by two residues belonging to the pIII
phage coat protein and at the C-terminal by a biotin moiety
For SPR analysis, peptides were immobilized on
strept-avidin-coated chips via their biotin moiety The selected
peptide was thus correctly exposed to the circulating
analytes The signal correponding to the immobilization
reaches 150–180 response units for each of the seven
peptides Binding of the rat anti-mouse IgG that was coated
in the selection procedure was assayed as the background
control Kinetic parameters and Kdvalues were determined
from sensorgrams and are reported in Table 4 IgGs being
dimeric proteins, the calculations were extrapolated from a
bivalent analyte model Among the seven assayed peptides,
three of them (Pep 91, Pep 93, Pep 95) do not significantly
bind to IgG 9G4H9 under SPR experimental conditions
Among the four other peptides, three (Pep 92, Pep 94 and
Pep 96) displayed a weakbinding ability not easily
meas-urable in SPR conditions, and one of them (Pep 90) revealed
a high affinity for the Ig, with a Kdvalue of 7.4· 10)8M
(Fig 4) As shown on the figure, the experimental data fit
the theoretical plot We verified the absence of mass transfer
interference by assaying several analysis conditions, notably
by varying the flow rate of the circulating analyte
Inhibition of the 9G4H9 b-lactamase activity
Pep 90 was tested for its inhibition properties of 9G4H9
catalytic activity Kinetics were carried out using peptide
concentrations from 0 to 180 lM Pep 95 was assayed as a negative control because it presents the same biochemical properties as Pep 90, notably its pI, but has a low affinity for 9G4H9 The inhibitory capabilities of Pep 90 were investigated by measuring the residual b-lactamase activity
of the IgG 9G4H9 after 20 min of incubation with various amounts of peptide (Fig 5) A half inhibitory concentration (IC50) can be calculated from this inhibition plot For 9G4H9, the IC50¼ 9.10)5M No inhibition effect could be measured with Pep 95 This indicates that the inhibitory effect is undoubtedly due to the selected sequence and not to biotin or phage pIII moieties
Strynadka et al have previously reported the character-ization of a 165-residue b-lactamase-inhibitor protein, BLIP (Ki¼ 0.3 nM) [13,14] We tested the inhibition of the b-lactamase activity of the anti-idiotypic Ig using a bacteriophage displaying BLIP on its surface, engineered and provided by T Palzkill, Dept of Molecular Virology and Microbiology, Baylor College of Medicine, Houston,
TX, USA [15] BLIP displayed on phage were found to inhibit the IgG with an IC50around 10)5M We then carried out a competitive ELISA assay, derived from the previously described phage ELISA, where IgG 9G4H9 was coated on
Table 4 Kinetic values and dissociation constants for seven selected
peptides Measurements were performed by SPR analysis BT, below
threshold; ND, not determined.
Peptide k on ( M )1 Æs)1) k off (s)1) K d ( M )
Pep 90 3.1 · 10 4 ± 4.8 · 10 2 2.3 · 10)3± 10)4 7.4 · 10)8
Pep 92 6.9 · 10 3
± 6.8 · 10 1
4.1 · 10)2± 4.8 · 10)3 5.9 · 10)6
Pep 94 4.6 · 10 3 ± 4.5 · 10 2 5.2 · 10)3± 5.5 · 10)4 1.1 · 10)6
Pep 96 4.8 · 10 3
± 7.1 · 10 1
1.2 · 10)3± 5.1 · 10)5 2.4 · 10)7
Fig 5 Inhibition of IgG 9G4H9 b-lactamase activity by Pep 90 9G4H9 (3.2 l M ) was incubated with various concentrations of Pep 90 (0–180 l M ) for 20 min at room temperature The activity was revealed with 1.8 m M ampicillin in phosphate buffer 0.1 M pH 7.4 (grey bar) Pep 95 (open bar) was used as a control from 0–100 l
Fig 4 Sensorgrams corresponding to SPR analysis Sensograms of SPR analysis of the binding of Pep 90 (40 lgÆmL)1in HBS) immo-bilized on SA chips, to various concentrations of IgG 9G4H9 from
156 n M to 468 n M , in HBS buffer, circulating at 7 lLÆmin)1 The experimental data fit the theoretical plot.
Trang 6plates and incubated with BLIP on phage The binding of
BLIP was measured by an anti-M13 peroxidase-conjugated
Ig, that gives a chromogenic signal in the presence of its
substrate We added various amounts of free Pep 90, from 0
to 100 lgÆmL)1 The residual binding of BLIP was then
estimated (Fig 6) Pep 90 was found to expel BLIP from
9G4H9 active site This clearly indicates that BLIP and
Pep 90 share the same interaction site
Discussion
The selection of inhibitory compounds is a real challenge
because biocatalysts are generally elaborate
macromole-cules We have developed a procedure to obtain peptides
that on the one hand display a tight affinity for the
biocatalyst and, on the other hand, valuably inhibit a
b-lactamase-like activity shown by an anti-idiotypic
cata-lytic Ig By this approach, we reduced the diversity of the
original library from more than one billion peptides to only
four, with one displaying the required function Our method
is all the more efficient in that the target, an IgG, is a
macromolecule of high molecular mass (approximately
150 kDa) thus displaying many potential interacting sites
We have previously suggested that only a few residues (two
serines, one lysine and one glutamic acid) were involved in
the catalytic activity of the Ig; they are located on the light
chain and gathered together [12,16]
It was previously demonstrated that cyclic peptides
displayed on phage could favourably lead to ligands
presenting better affinities than linear ones [17] However,
the main restriction in the development of procedures based
on phage display technology to obtain worthwhile
com-pounds is that the selected molecules are interesting only if
they retain their activity in their soluble form Some authors
indicate that cyclic peptides are more likely to retain their
function once in free form than their linear counterparts
[18] In our work, four peptides out of seven have retained
binding features once in soluble form
The sequence of the inhibitory peptide Pep 90
Tyr-His-Phe-Leu-Trp-Gly-Pro is mainly composed of central
hydro-phobic residues and contains a Pro in the C-terminal,
similar to most of the peptides selected against IgG 9G4H9
This result allows the definition of a basic feature of
structures able to bind to this IgG active site BLIP was described to be an efficient inhibitory protein of b-lactamase [13] and it was found to also inhibit IgG 9G4H9 with a weaker IC50 The residues of BLIP that are involved in the inhibition function of b-lactamase were identified [19,20] as Ala-Ala-Gly-Asp-Tyr-Tyr, which form a hairpin The primary structures of this part of BLIP and Pep 90 are different, and their tri-dimensional schemes cannot be overlaid Consequently, the selection procedures that we have developed have led us to find a completely new solution for the inhibition of a catalyst
The ability of BLIP to inhibit IgG 9G4H9 is a crucial point because it proves that the active site IgG 9G4H9 is the real internal image of b-lactamase The b-lactamase-like catalytic information was then successfully transferred from the enzyme to the abzyme throughout the idiotypic pathway The catalytic features of a biocatalyst, such as activity and inhibition by elaborated molecules, can thus be preserved by the immune system within the idiotypic network This opens routes towards the idea that auto-immune diseases involving catalytic antibodies [21,22] might have appeared because of the presence of abnormal amounts of nonimmunogen molecules stimulating the apparition of catalytic Igs
The procedure developed is efficient for the selection of inhibitory peptides even in the absence of any information about the structure of the target It is much less time-consuming and less expensive than rationally designed synthetic compounds Indeed, b-lactamase is widespread throughout the world and is considered to be a plague for the treatment of infections by antibiotics [23] Designing new antibiotic molecules is a flourishing activity of medical research nowadays The in vitro selection of peptides able to inhibit b-lactamase activity appears, consequently, as a good alternative route to the rational design of small molecules based on the knowledge of the targeted catalytic mechanisms
Acknowledgements
We are grateful to Drs Barbee and Koivunen for kindly providing the peptide library and Dr Palzkill for giving its BLIP construction for the expression on phage We gratefully acknowledge the Picardie region and ALTERNATECH for supporting A.S Yribarren.
References
1 Azzazy, H.M.E & Highsmith, W.E Jr (2002) Phage display technology: clinical applications and recent innovations Clin Biochem 35, 425–445.
2 Smith, G.P (1985) Filamentous fusion phage: novel expression vectors that displayed cloned antigens on the virion surface Science 228, 1315–1317.
3 Parmley, S.F & Smith, G.P (1988) Antibody-selectable fila-mentous fd phage vectors: affinity purification of target genes Gene 73, 305–318.
4 Devlin, J.J., Panganiban, L.C & Devlin, P.E (1990) Random peptide libraries: a source of specific binding molecules Science
249, 404–406.
5 Avalle, B., Friboulet, A & Thomas, D (2000) Catalysis by anti-idiotypic antibodies Chem Immunol 77, 80–88.
6 Kolesnikov, A.V., Kozyr, A.V., Alexandrova, E.S., Koralewski, F., Demin, A.V., Titov, M.I., Avalle, B., Tramontano, A., Paul, S.,
Fig 6 Competitive ELISA for the binding to IgG 9G4H9 Phage BLIP
(1011TU) were incubated on 1 lg coated IgG 9G4H9 After 1 h
incubation at 37 C, various amounts of Pep 90 (0–100 l M ) were
added to wells Absorbance values are related to the residual binding of
BLIP to IgG 9G4H9 The CX 7 C library and M13 were assayed to
evaluate the background binding signal The standard deviations were
calculated from two separate experiments.
Trang 7Thomas, D., Gabibov, A.G & Friboulet, A (2000) Enzyme
mimicry by the anti-idiotypic antibody approach Proc Natl Acad.
Sci USA 97, 13526–13531.
7 Avalle, B., Thomas, D & Friboulet, A (1998) Functional
mimicry: elicitation of a monoclonal anti-idiotypic antibody
hydrolysing b-lactams FASEB J 11, 1055–1060.
8 Pillet, D., Paon, M., Vorobiev, I.I., Gabibov, A.G., Thomas, D &
Friboulet, A (2002) Idiotypic networkmimicry and antibody
catalysis: lessons for the elicitation of efficient anti-idiotypic
pro-tease antibodies J Immunol Methods 269, 5–12.
9 Jerne, N.K (1974) Toward a networktheory of the immune
system Ann Immunol 125c, 373–389.
10 Avalle, B., Mistro, D., Thomas, D & Friboulet, A (1996)
Poly-clonal catalytic anti-idiotypic antibodies with b-lactamase activity.
Ann NY Acad Sci 799, 172–175.
11 Koivunen, E., Wang, B & Ruoslahti, E (1994) Isolation of a
highly specific ligand for the alpha-5, beta-1 integrin from a phage
display library J Cell Biol 124, 373–380.
12 Lefe`vre, S., De´bat, H., Thomas, D., Friboulet, A & Avalle, B.
(2001) A suicide substrate mechanism for hydrolysis of b-lactams
by an anti-idiotypic catalytic antibody FEBS Lett 489, 25–28.
13 Strynadka, N.C., Jensen, S.E., Johns, K., Blanchard, H., Page,
M., Matagne, A., Frere, J.M & James, M.N (1994) Structural
and kinetic characterization of a beta-lactamase-inhibitor protein.
Nature 368, 657–660.
14 Strynadka, N.C., Jensen, S.E., Alzari, P.M & James, M.N (1996)
A potent new mode of beta-lactamase inhibition revealed by the
1.7 A X-ray crystallographic structure of the TEM-1-BLIP
com-plex Nat Struct Biol 3, 290–297.
15 Huang, W., Zhang, Z & Palzkill, T (2000) Design of potent beta-lactamase inhibitors by phage display of beta-beta-lactamase inhibitory protein J Biol Chem 275, 14964–14968.
16 De´bat, H., Avalle, B., Friboulet, A & Thomas, D (2000) Structure-function studies of a new generation of catalytic protein:
an abzyme with a b-lactamase activity Int J Biochromat 5, 91–96.
17 Giebel, L.B., Cas, R.T., Milligan, D.L., Young, D.C., Arze, R & Johnson, C.R (1995) Screening of cyclic peptide phage libraries identifies ligands that bind streptavidin with high affinities Biochemistry 34, 15430–15435.
18 McLafferty, M.A., Kent, R.B., Ladner, R.C & Markland, W (1993) M13 bacteriophage displaying disulfide constrained microproteins Gene 128, 29–36.
19 Rudgers, G.W., Huang, W & Palzkill, T (2001) Binding properties of a peptide derived from beta-lactamase inhibitory protein Antimicrob Agents Chemother 45, 3279–3286.
20 Rudgers, G.W & Palzkill, T (2001) Protein minimization by random fragmentation and selection Protein Eng 14, 487–492.
21 De´bat, H., Avalle, B., Chose, O., Sarde, C.O., Friboulet, A & Thomas, D (2001) Overpassing an aberrant Vkgene to sequence
an anti-idiotypic abzyme with b-lactamase like activity that could have a possible linkage with autoimmune disease FASEB J 15, 815–822.
22 Friboulet, A., Avalle, B., De´bat, H & Thomas, D (1999) A possible role of catalytic antibodies in metabolism Immunol Today 20, 474–475.
23 Helfand, M.S & Bonomo, R.A (2003) Beta-lactamases: a survey
of protein diversity Curr Drug Targets Infect Disord 3, 9–23.