Toward a Better Understanding of the Basis of the Molecular Mimicry of Polysaccharide Antigens by Peptides

Abstract

Protein conjugates of oligosaccharides or peptides that mimic complex bacterial polysaccharide antigens represent alternatives
to the classical polysaccharide-based conjugate vaccines developed so far. Hence, a better understanding of the molecular
basis ensuring appropriate mimicry is required in order to design efficient carbohydrate mimic-based vaccines. This study
focuses on the following two unrelated sets of mimics of the Shigella flexneri 5a O-specific polysaccharide (O-SP): (i) a synthetic branched pentasaccharide known to mimic the average solution conformation
of S. flexneri 5a O-SP, and (ii) three nonapeptides selected upon screening of phage-displayed peptide libraries with two protective murine
monoclonal antibodies (mAbs) of the A isotype specific for S. flexneri 5a O-SP. By inducing anti-O-SP antibodies upon immunization in mice when appropriately presented to the immune system, the
pentasaccharide and peptides p100c and p115, but not peptide p22, were qualified as mimotopes of the native antigen. NMR studies
based on transferred NOE (trNOE) experiments revealed that both kinds of mimotopes had an average conformation when bound
to the mAbs that was close to that of their free form. Most interestingly, saturation transfer difference (STD) experiments
showed that the characteristic turn conformations adopted by the major conformers of p100c and p115, as well as of p22, are
clearly involved in mAb binding. These latter experiments also showed that the branched glucose residue of the pentasaccharide
was a key part of the determinant recognized by the protective mAbs. Finally, by using NMR-derived pentasaccharide and peptide
conformations coupled to STD information, models of antigen-antibody interaction were obtained. Most interestingly, only one
model was found compatible with experimental data when large O-SP fragments were docked into one of the mIgA-binding sites.
This newly made available system provides a new contribution to the understanding of the molecular mimicry of complex polysaccharides
by peptides and short oligosaccharides.

Full text

Page 1

Toward a Better Understanding of the Basis of the Molecular
Mimicry of Polysaccharide Antigens by Peptides
THEEXAMPLEOFSHIGELLAFLEXNERI5A*□
Receivedforpublication,September15,2005,andinrevisedform,October25,2005 Published,JBCPapersinPress,October26,2005,DOI10.1074/jbc.M510172200
Marie-Jeanne Cle ´ment‡, Antoine Fortune ´§, Armelle Phalipon¶, Ve ´ronique Marcel-Peyre¶, Catherine Simenel‡,
Anne Imberty?, Muriel Delepierre‡1, and Laurence A. Mulard**
Fromthe‡Unite ´ deRMNdesBiomole ´cules,URACNRS2185,InstitutPasteur,**Unite ´ deChimieOrganique,URACNRS2128,Institut
Pasteur,¶Unite ´ dePathoge ´nieMicrobienneMole ´culaire,InstitutPasteur,28RueduDr.Roux,75724ParisCedex15,
§DPMUMR5063UJF/CNRS,5AvenuedeVerdun38240Meylan,France,andthe?CERMAV-CNRS(affiliatedwith
Universite ´ JosephFourier),38041 Grenoble BP53, Cedex 09, France
S
Protein conjugates of oligosaccharides or peptides that mimic
complexbacterialpolysaccharideantigensrepresentalternativesto
the classical polysaccharide-based conjugate vaccines developed so
far. Hence, a better understanding of the molecular basis ensuring
appropriate mimicry is required in order to design efficient carbo-
hydrate mimic-based vaccines. This study focuses on the following
two unrelated sets of mimics of the Shigella flexneri 5a O-specific
polysaccharide (O-SP): (i) a synthetic branched pentasaccharide
known to mimic the average solution conformation of S. flexneri 5a
O-SP,and(ii)threenonapeptidesselecteduponscreeningofphage-
displayed peptide libraries with two protective murine monoclonal
antibodies (mAbs) of the A isotype specific for S. flexneri 5a O-SP.
By inducing anti-O-SP antibodies upon immunization in mice
whenappropriatelypresentedtotheimmunesystem,thepentasac-
charide and peptides p100c and p115, but not peptide p22, were
qualifiedasmimotopesofthenativeantigen.NMRstudiesbasedon
transferred NOE (trNOE) experiments revealed that both kinds of
mimotopes had an average conformation when bound to the mAbs
that was close to that of their free form. Most interestingly, satura-
tiontransferdifference(STD)experimentsshowedthatthecharac-
teristic turn conformations adopted by the major conformers of
p100candp115,aswellasofp22,areclearlyinvolvedinmAbbind-
ing. These latter experiments also showed that the branched glu-
cose residue of the pentasaccharide was a key part of the determi-
nant recognized by the protective mAbs. Finally, by using NMR-
derived pentasaccharide and peptide conformations coupled to
STD information, models of antigen-antibody interaction were
obtained.Mostinterestingly,onlyonemodelwasfoundcompatible
with experimental data when large O-SP fragments were docked
into one of the mIgA-binding sites. This newly made available sys-
temprovidesanewcontributiontotheunderstandingofthemolec-
ular mimicry of complex polysaccharides by peptides and short
oligosaccharides.
Bacterial capsular polysaccharides (CPS)2and lipopolysaccharides
(LPS) are known to be important virulence factors and major targets of
the protective immune response of the host (1). Several polysaccharide
vaccines such as those targeting Streptococcus pneumoniae, Neisseria
meningitidis,orSalmonellatyphiwereprovenefficientinadultsandare
thus commercially available. Their ineffectiveness in infants has been
successfully circumvented with the licensing of polysaccharide:protein
conjugates such as those targeting Haemophilus influenzae b, S. pneu-
moniae, and N. meningitidis group C infections (2). A possible alterna-
tive may derive from the use of accurate synthetic mimics of the bacte-
rial polysaccharide antigens. This innovative approach has been mostly
developed along two lines, including the use of either synthetic oligo-
saccharides or peptides mimicking the carbohydrate determinants rec-
ognizedbyanti-carbohydratemonoclonalantibodies(mAb)conferring
protection in experimental models of infection. Indeed, semi-synthetic
glycoconjugates incorporating oligosaccharides mimicking fragments
of bacterial polysaccharide antigens were shown to be highly immuno-
genic in mice (3–5). The “proof of concept” was recently demonstrated
in humans with the efficacy of such a semi-synthetic glycoconjugate in
protecting against H. influenzae b infection (6).
However,accesstotherequiredcarbohydratehaptensisoftenaroad-
block.Therefore,besidestheinvestigationofanti-idiotypeantibody(7),
expanding the concept of mimicry led in the recent past to extensive
exploringofthepotentialmimickingofpolysaccharideand/orcomplex
oligosaccharideantigensbypeptides(8–10).Thesepeptidemimotopes,
i.e. peptide mimics inducing an anti-carbohydrate antibody response
upon immunization, have been proposed as potential surrogate anti-
gens of carbohydrates in vaccine development (10). Indeed, because of
their ease of manufacture and their intrinsic immunogenic properties,
peptidemimotopesmayhavegreateradvantageovercomplexcarbohy-
drate haptens issued from bacterial cell cultures or low yielding multi-
step syntheses. However, not all peptide mimics of carbohydrate anti-
gens behave as mimotopes. Despite the large number of known peptide
mimics, only few peptide mimotope-based experimental vaccines have
been reported so far (11–16).
It is believed that a better understanding of the molecular basis of
peptide-carbohydrate mimicry could help the rational design of potent
peptide mimotope-based vaccines. In particular, whether mimicry is
* This work was supported by MENRT (Programme de Recherche Fondamentale en
Microbiologie et Maladies Infectieuses et Parasitaires), De ´le ´gation Ge ´ne ´rale pour
l’ArmementContract9934029,andtheCNRSProgramPhysiqueetChimieduVivant.
The costs of publication of this article were defrayed in part by the payment of page
charges.Thisarticlemustthereforebeherebymarked“advertisement”inaccordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
□
SThe on-line version of this article (available at http://www.jbc.org) contains Figs.
1S–4S.
1Towhomcorrespondenceshouldbeaddressed:Unite ´ deRMNdesBiomole ´cules,Insti-
tutPasteur,28RueduDr.Roux,75724ParisCedex15,France.Tel.:33-1-45-68-88-71;
Fax: 33-1-45-68-89-29; E-mail: murield@pasteur.fr.
2The abbreviations used are: CPS, capsular polysaccharides; NOE, nuclear Overhauser
effect;tr,transferred;ROESY,rotatingframenuclearOverhauserenhancementspec-
troscopy; NOESY, nuclear Overhauser effect spectroscopy; ELISA, enzyme-linked
immunosorbent assay; Ab, antibody; mAb, monoclonal Ab; STD, saturation transfer
difference; O-SP, O-specific polysaccharide; LPS, lipopolysaccharides; PBS, phos-
phate-buffered saline; BSA, bovine serum albumin; TOCSY, total correlation
spectroscopy.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 4, pp. 2317–2332, January 27, 2006
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.Printed in the U.S.A.
JANUARY 27, 2006•VOLUME 281•NUMBER 4 JOURNAL OF BIOLOGICAL CHEMISTRY 2317

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structural,functional,orbothremainsanunsolvedquestion(9,17–19).
Available x-ray data of carbohydrate-protein and of the corresponding
peptide mimotope-protein complexes along with information on the
thermodynamics of peptide mimic-protein binding are somewhat
scarce (17, 20, 21). Thus to date, although analysis of the topography of
ligand-receptor complementarity may be performed by a variety of
methods, available knowledge on the molecular features of peptide-
carbohydrate mimicry mostly relies on data obtained from NMR and
molecular modeling studies as reviewed recently (22).
By aiming to prevent S. flexneri bacterial infections, during the past
few years we investigated the development of synthetic mimics of the
majorprotectiveS.flexneriantigen.S.flexneri,aGram-negativebacillus,
is responsible for the endemic form of shigellosis, a human dysenteric
syndrome causing a high mortality rate in infants, particularly in devel-
opingcountries(23).Thedisease,characterizedbybacterialinvasionof
the human colonic mucosa, leads to acute inflammation and subse-
quent massive tissue destruction (24). Protection induced upon infec-
tion is serotype-specific (24), pointing to the O-specific polysaccharide
moiety (O-SP) of the bacterial LPS as the major target for protective
immunity. In line with the success of the CPS-protein conjugate vac-
cines, protein conjugates of detoxified S. flexneri 2a LPS, the prevalent
serotype in humans, were shown to be safe and immunogenic both in
adults and young children (25). More recently, we developed fully syn-
thetic glycoconjugates as well as promising neoglycoproteins exposing
well designed synthetic saccharidic haptens mimicking S. flexneri 2a
O-SP as potential vaccines against the homologous infection (26, 27).3
Alternatively, we also investigated the potential of peptide mimotopes,
and we reported the first example of immunogenic mimicry of carbo-
hydrates by peptides identified by screening of phage-displayed non-
apeptide libraries with two protective mAbs of the A isotype (mIgA)
specific for S. flexneri serotype 5a, mIgA C5, and mIgA I3 (28). Among
the 19 peptide sequences selected upon screening with mIgA I3, p100c
(YKPLGALTH) and p115 (KVPPWARTA, also interacting with mIgA
C5) only induce anti-O-SP antibodies in mice upon immunization with
the corresponding phage particles. Most interestingly, the mimotopes
share no obvious consensus sequence and do not cross-react with one
another.However,asoftenreportedbyothers(29,30),theiraminoacid
sequences contain aromatic and hydrophobic residues but also amino
acids having cyclic side chains, including at least one proline.
Besides, based on a combination of NMR and molecular modeling
studies,weproposedaconformationalmodelfortheS.flexneri5aO-SP
whose biological repeating unit is the branched pentasaccharide I
(Structure 1) (31). Study of both the antigenicity and the conformation
of the four synthetic frame-shifted pentasaccharides corresponding to
pentasaccharide I (32) suggested that the DA(E)BC sequence is the
structure that best mimics the native O-SP antigen (33). More recently,
the pentasaccharide DA(E)BC was shown to act as a mimotope.4
Here we report the antigenicity and the NMR findings on the pre-
ferredconformationofp100candp115peptidemimotopesbothintheir
freeandmIgA-boundforms.Analysiswasalsoperformedusingpeptide
p22 (KRHFLSQRQ, mIgA C5- and mIgA I3-specific), one of the 17
nonimmunogenic peptides selected during the original screening (28).
Antibody-bound conformations and epitope mapping were derived
from transferred NOE (trNOE) (34, 35) and saturation transfer differ-
ence (STD) experiments (36), respectively. The conformational prefer-
encesobservedforthepeptidesweretentativelyrelatedtothosederived
from NMR and molecular modeling analysis of the DA(E)BC-mIgA
complexesthatledtoatheoreticalmodeloftherecognitionofS.flexneri
5a O-SP by mIgA I3. This contribution adds to the few reports investi-
gating molecular mimicry by analyzing both peptide mimic-mAb and
carbohydrate-mAb recognition features (37–39).
EXPERIMENTAL PROCEDURES
Material—Selected nonapeptides p100c (YKPLGALTH), p115
(KVPPWARTA), and p22 (KRHFLSQRQ) were purchased from Syn-
them (Saint-Christol-Les-Ale `s, France). The peptide p100c was further
cyclized in the Unite ´ de Chimie Organique at the Pasteur Institute.
Pentasaccharide DA(E)BC was used in its methyl glycoside form
DA(E)BC-OMe (40). mAb mIgA C5 and mIgA I3 were prepared as
described previously (41).
Inhibition ELISA—Characterization of the oligosaccharide determi-
nant recognized by the mIgA was performed by measuring the mIgA-
oligosaccharide interaction as follows. First of all, a standard curve was
established for each mIgA tested. Different concentrations of the mAb
were incubated overnight at 4 °C on microtiter plates coated with puri-
fiedS.flexneri5aLPSataconcentrationof5 ?g/mlincarbonatebuffer,
pH9.6,andsubsequentlyincubatedwith1%PBS/BSAfor30minat4 °C.
After washing with PBS/Tween 20 (0.05%), alkaline phosphatase-con-
jugatedanti-mouseIgAwasaddedatadilutionof1:5,000(Sigma)for1h
at 37 °C. After washing with PBS/Tween 20 (0.05%), the substrate was
added (12 mg of p-nitrophenyl phosphate in 1.2 ml of 1 M Tris-HCl
buffer, pH 8.8, and 10.8 ml of 5 M NaCl). Once the color developed, the
plate was read at 405 nm (Dynatech MR 4000 microplate reader). A
standard curve A ? f([Ab]) was fitted to the quadratic equation Y ?
aX2?bX?c,whereYistheabsorbanceandXistheAbconcentration.
Correlation factor (r2) of 0.99 was routinely obtained.
Then the amount of oligosaccharides giving 50% inhibition of mIgA
binding to LPS (IC50) was determined as follows. Each mIgA at a given
concentration (chosen as the minimal concentration of Ab which gives
the maximal absorbance on the standard curve) was incubated over-
nightat4 °Cwithvariousconcentrationsofeachoftheoligosaccharides
to be tested, in 1% PBS/BSA. Measurement of unbound mIgA was per-
formed as described above using microtiter plates coated with purified
LPS from S. flexneri 5a, and the mAb concentration was deduced from
the standard curve.
The recognition capacity of anti-LPS mIgA for LPS was determined
as described above using various concentrations of LPS that were incu-
batedinsolutionovernightat4 °Cwiththepredefinedconcentrationof
each mIgA. IC50was defined as the concentration of oligosaccharides
required to inhibit 50% of mIgA binding to LPS.
NMR Spectroscopy—All1H NMR experiments were recorded at 298
K on a Varian Unity Inova spectrometer operating at1H frequencies of
500MHz.1Hchemicalshiftsweregivenrelativetoanexternalstandard
of 4,4-dimethyl-4-silapentane sodium sulfonate at 0 ppm.
FreePeptides—Thesampleswerepreparedin90%H2Oand10%D2O
at pH 5 for p115 and p100c and at pH 6.5 for p22. The solution concen-
trations were about 10, 3, and 8 mM for p115, p100c, and p22, respec-
tively. DQF-COSY (42), TOCSY (43), and ROESY (44) experiments
were recorded with 512 increments and 16 scans at 298 K. The TOCSY
3Phalipon,A.,Costachel,C.,Grandjean,C.,Thuizat,A.,Guerreiro,C.,Tanguy,M.,Nato,F.,
Vulliez-Le Normand, B., Be ´lot, F., Wright, K., Marcel-Peyre, V., Snsonetti, P. J., and
Mulard, L. (2005) J. Immunol., in press.
4L. Mulard and A. Phalipon, unpublished results.
STRUCTURE 1
PeptideMimicsofS.flexneri5aPolysaccharideAntigen
2318 JOURNAL OF BIOLOGICAL CHEMISTRYVOLUME 281•NUMBER 4•JANUARY 27, 2006

Page 3

and ROESY experiments were acquired using a mixing time of 80 and
400 ms, respectively. Water suppression was performed using the
WATERGATE pulse sequence (45). All NMR spectra were collected in
the phase-sensitive mode using the States-Haberkorn method (46).
Ligand-Antibody Interactions—Shigemi tubes were used for all sam-
ples. In order to prepare NMR samples of pentasaccharide DA(E)BC-
OMe in the presence of the antibodies mIgA C5 and mIgA I3, mAbs
were concentrated after repeated cycles of exchange with D2O buffer
(50mMdeuteratedsodiumphosphate,100mMNaCl,pH6.5)inAmicon
Centriprep-10 concentrators. trNOE experiments (34, 35, 47) per-
formed on different pentasaccharide:binding site ratios (5:1, 10:1, 15:1,
20:1,and30:1)showedthatthemostfavorableratiofortrNOEwas20:1.
So the final samples were prepared with 3.75 ?M antibody and 0.3 mM
pentasaccharide in 380 ?l of the above mentioned D2O buffer. trNOE,
trROE, and STD experiments (36) on pentasaccharide DA(E)BC-OMe
in the presence of mIgA C5 and mIgA I3 were recorded at 500 and 600
MHz, respectively. trNOE experiments were performed with mixing
times of 100, 150, 250, 300, and 400 ms at 303 K to obtain build-up
curves and trROE with a mixing time of 400 ms.
TheconformationofthefreepeptideswasstudiedatpH5.However,
with this pH value being close to the isoelectric points of the mIgAs, a
studyofpeptidesintheirboundconformationwasperformedatpH6.5
to avoid precipitation of the mAb. Similarly to the DA(E)BC-mIgA
complexes, a peptide:antibody-binding site ratio of 20:1 was used (0.3
mM:3.75?M).trNOEexperimentswereperformedwithmixingtimesof
100, 150, 250, 300, and 400 ms. To be sure that the observed negative
cross-peakswererealtrNOEs,NOESYspectrawererecordedunderthe
same pH, temperature, and concentration values with the peptides
alone. Furthermore, to discard any impact on NOE effects of viscosity
increase as a result of the mAb presence, a NOESY spectrum (?m? 200
ms) of p115 was registered in the presence of BSA at the same concen-
tration ratio as that used with the mIgAs. Because no negative NOE
cross-peaks were observed in either case, it was assumed that the neg-
ative NOEs observed in the presence of mIgA were trNOEs.
Selective saturation of antibody resonances were performed for all
STD-NMRexperimentsat0.3ppm(30ppmforreferencespectra)using
a series of 40 gaussian-shaped pulses (50- and 10-ms delay between
pulses, excitation width ?B1/2?, approximately 50 Hz) for a total satu-
ration time of 2.4 s. The one-dimensional STD spectra were recorded
with 4096 scans at 288 and 298 K for the pentasaccharide and the pep-
tides, respectively. Subtraction of saturated spectra from reference
spectra was obtained by phase cycling (36). For DA(E)BC-OMe, two
STD-TOCSY experiments (48) were recorded with selective saturation
at 0.3 and 30 ppm, respectively. Differences between the two spectra
were performed using the VNMR software. No attempt here was made
to quantify STD-NMR intensities, as it is known that these exhibit a
complex dependence on relaxation times, correlation times, exchange
rates, and on binding site proton density. Indeed, only when short sat-
uration times are used, i.e. less than 1 s, can intensities reflect ligand
proton-protein proton distances (37). Here the saturation time of 2.4 s
prevented us from quantitative analysis.
Distance and Angle Constraints—The cross-peak volumes from
trNOESYandtrROESYexperimentsofthepentasaccharideinthepres-
ence of mIgAs were measured with the VNMR software. Distances
between neighboring protons were calculated by the usual 1/r6NOE/
distance relationship (49). NOE-derived and trROE distances were
obtained from initial NOE build-up rates, which were calculated by
NOE volumes fitting during different mixing times. The intra-residue
distance of 2.52 Å between the H-1 and H-2 protons of the ?-L-rham-
nopyranosyl unit B was used as a reference for distance calibration.
Distance constraints of free peptides were obtained from the ROESY
spectrum run at 298 K with a 400-ms mixing time. For peptides in the
presence of mIgA, distance constraints were obtained from the
trNOESY spectra run at 298 K with a 200-ms mixing time. NOE inten-
sities were evaluated from the height of the cross-peaks. For structure
calculations, upper limit distances of 2.8, 3.5, and 5 Å were used for
strong, medium, and weak NOEs, respectively (50). The3JNH-H?values
were used to restrain ? angles as follows: for3J ?9 Hz, ?155° ? ? ?
?85°; for 8 Hz ?3J ?9 Hz, ?175° ? ? ? ?65°; for 5 Hz ?3J ?7 Hz,
?105° ? ? ? ?55°; for3J ?5 Hz, ?90° ? ? ? ?40° (51).
Structure Calculations—Structure calculations of free and bound
peptideswererunonaSiliconGraphicsworkstationusingthestandard
protocol of the DYANA program (52). A total of 100 structures were
calculated using the torsion angle dynamics protocol. The structures
weresortedaccordingtothefinalvalueofthetargetfunction,andthe20
best structures were analyzed in terms of distance and angle violations.
Of these 20 structures, the 10 best structures were visualized by using
MOLMOL (53).
Homology Modeling of the IgA I3 Fab Fragment and Docking—The
search for structures with sequences similarities was performed with
Blast (54) on sequences of all proteins with known three-dimensional
structure in the Protein Data Bank (55). Five structures of interest were
downloaded and used as template by the Composer program for the
building of VL and VH chains of IgA I3 (56).
The Tripos force field (57) option of the Sybyl program (SYBYL) was
used to minimize the energy of the resulting model whose stereochem-
ical features were validated with the PROCHECK program (58).
The Autodock3 program (59) was used for docking oligosaccharides
andpeptidesinthebindingsiteofmodeledIgAI3Fab.Becausethegoal
wastomodelthebehavioroftheO-SP,calculationswereperformedon
the largest possible fragment compatible with the limitations of the
software, in that case a nonasaccharide. The 9-carbohydrate residue
fragment was thus chosen as BCDA(E)BCDA in which the key pen-
tasaccharide DA(E)BC is flanked by two residues on each side. The two
conformations that were shown previously to correspond to helical
shapes of the O-SP (33) were used as starting models. Hydroxyl and
N-acetyl bonds were considered as flexible, whereas glycosidic bonds
were considered as rigid to keep the helical conformation, resulting in
28 degrees of freedom. AMBER force field charges were assigned to all
proteinatoms,andpartialchargeswereassignedtotheatomsaccording
tothePIMforcefield(60).Gridsofprobeatominteractionenergiesand
electrostatic potential were generated around the whole protein by the
AutoGrid program present in Autodock3 with a spacing of 0.5 Å. All
probes were placed arbitrarily at a distance of 10 Å from the protein
surface, and their exocyclic torsion angles were allowed to rotate freely.
For each monosaccharide, one job of 240 docking runs was performed
using a population of 100 individuals and an energy evaluation number
of10?106.Clusteringofsolutionswasdonebyrootmeansquarefitting
(?1 Å). The best solution of each cluster was used to propagate the
helicesto20residueswhilekeepingtheconformationsdeterminedpre-
viously (33). Twenty different conformers of the p100c peptide were
also docked in the mIgA I3 Fab-binding site using the rigid body
approachoftheAutodock3program.Foreachofthem,onedockingrun
was performed using a population of 100 individuals and an energy
evaluation number of 0.75 ? 106.
RESULTS
Antigenicity of the Ligands Used in the Study—The binding of the
synthetic nonapeptides p100c, p115, and p22 (28) and synthetic pen-
tasaccharide DA(E)BC-OMe (32) to mIgA I3 and mIgA C5 was evalu-
PeptideMimicsofS.flexneri5aPolysaccharideAntigen
JANUARY 27, 2006•VOLUME 281•NUMBER 4JOURNAL OF BIOLOGICAL CHEMISTRY 2319

Page 4

ated by inhibition ELISA to determine the concentration of ligands
inhibiting 50% of mIgA binding to LPS (IC50value). Because of the
multivalency of both the mIgAs (dimeric mAb, thus four binding sites)
and LPS, the IC50value does not reflect the true binding affinity but
allows relative comparison of the ligand recognition by the mIgAs. In
agreement with the low affinity of mAb for carbohydrate antigens, an
IC50valuecloseto25mMwasobservedfortheinteractionofDA(E)BC-
OMe with each of the mIgAs.
IC50values for recognition of the mimotopes by mIgAs revealed that
p100c was better recognized by mIgA I3 (IC50? 75 ? 29 ?M) than by
mIgA C5 (IC50?1000 ?M). In contrast, p115 was better recognized by
mIgAC5(IC50?197?39?M)thanbymIgAI3(IC50?1000?M).Most
interestingly,p22exhibitedahigherIC50valueforbothmIgAs(70?11
?M and 0.03 ? 0.01 ?M for mIgA I3 and mIgA C5, respectively) than
those measured for p100c and p115.
NMR Parameters for the Free Peptide—Peptide proton chemical
shifts were assigned following standard procedures (50). The peptide
conformations were probed through analysis of proton chemical shifts,
three bond
interactions observed in the ROESY spectra. Dihedral angles and dis-
tance constraints deduced from these data were then used to model the
averaged solution structure of each peptide analyzed with the DYANA
program (52).
Peptide 115 (KVPPWARTA)—The NMR spectrum revealed that
p115 displayed four different conformers as a result of the cis-trans
isomerizationoftheamidebondsinvolvingthetwosequentialprolines,
Val2–Pro3and Pro3–Pro4, respectively. Based on signal intensities, it
was estimated that the major conformer represented 80% of the differ-
ent species, whereas the three other forms altogether made up for the
remaining20%.Becausenoinformationwasavailablefortheconformer
recognized upon selection from the phage displayed peptide library,
structuralanalysiswasconductedforthismajorconformeronly.Chem-
ical shifts and three bond3JNH-H?coupling constants are reported in
Table 1. Significant deviations from random coil values are only
observed for the H-? protons of Pro3and Pro4, whereas all three bond
3JNH-H?coupling constants are those expected for flexible peptides.
Inter-residue dipolar interactions observed in the ROESY experiment
between the H-? of the residue preceding a proline and the H-? proton
of the proline indicates that both Pro3and Pro4adopt a trans-confor-
mationinthepeptidemajorconformer.Inadditiontostandardsequen-
tial interactions, several medium range interactions were also observed
between side chain protons of Val2, Pro3, and Pro4and the CH3-? pro-
tons of Ala6(Fig. 1). These ROE connectivities were used as distance
constraints to model the conformation of the p115 major conformer
using the DYANA program. The 10 best structures, i.e. with the lowest
3JNH-H?coupling constants, and proton-proton dipolar
TABLE1
1H chemical shifts of the trans-trans-isomer of p115 in H2O/D2O (90/
10), pH 5.1 and 298 K
Chemical shifts measured in ppm with an accuracy of ?0.01 ppm are referenced to
external 4,4-dimethyl-4-silapentane sodium sulfonate (?H0.00).
Residue
Lys1
4.02
?0,30b
HN
NDa
H?
H?
1.84
Others
H-?1.38
H-?1.66
H-?2.96
H-?
H-?0.94–0.89
H-?1.71–1.85
H?3.81–3.51
H-?1.99
H-?3.69–3.44
H-?17.24
H-?37.16
H-?110.26
ND
Val2
Pro3
8.54 (ND)4.41
4.52
?0,21
4.28
?0,14
4.61
?0,05
2.02
1.97–1.05
Pro4
2.24–1.91
Trp5
7.31 (6.80)c
3.35–3.26
Ala6
7.64 (6.60) 4.27
?0,05
4.28
?0,06
1.15
Arg7
8.00 (6.70) 1.82–1.60 H-?1.72
H-?3.17
H-?7.16–6.65
H-?1.17
Thr8
8.17 (8.10)4.30
?0,05
4.11
4.23
Ala9
7.97 (6.60)1.32
aND indicates not determined.
bData in italics are the difference between the H? chemical shifts of the residues of
thepeptideandthoseofthesameresiduesinnonstructuredpeptidesGGXAGGor
GGXPGG (90).
cData in parentheses are3JHN,H?coupling constants (in Hz ? 0.2 Hz) measured
from the one-dimensional spectrum.
FIGURE1.1H-11HNOEconnectivitiesobservedforthemajorconformersofpeptides.A,p115trans-trans-isomer;B,p100ctrans-isomer;C,p22.TheintensityoftheNOEcross-peak
is indicated by the thickness of the lines (weak, –; medium, O; strong f).
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energyfunction,showedthatp115adoptsaratherorganizedconforma-
tion comprising residues Pro3to Arg7, whereas the N- and C-terminal
endsarequiteflexible(Fig.2)asexpectedforpeptidesofthissize.Based
on C-?i–C-?i?3distances as well as on ? and ? angle values (Table 2),
the conformation of the Pro3–Arg7fragment can be described as two
sequential?-turns,anonclassifiedoneforPPWAandatypeI?-turnfor
PWAR (61, 62).
Peptide 100c (CYKPLGALTHC)—Selected from a library displaying
nonapeptides flanked by two cysteines (pVIII-9aa Cys) (28), synthetic
p100c was chemically converted into its cyclic form. NMR data showed
that in solution p100c existed as a 9:1 equilibrium between two con-
formers resulting from the cis-trans-isomerization of the amide bond
between Lys3and Pro4. Again, despite the lack of information on the
p100c-mIgA I3 recognition, only the major conformer was analyzed
(Table 3). Significant deviation from standard chemical shift values was
observedfortheH-?protonsofresiduesTyr2,Lys3,Pro4,Leu5,andGly6
suggesting some restricted flexibility along the Tyr2–Gly6sequence.
Furthermore, the three bond3JNH-H-?coupling constant values for res-
idues Leu5, Gly6, and Ala7are slightly smaller than those measured for
the other residues, 5 Hz versus 7–8 Hz (Table 3), strengthening the
hypothesis of a probable structuring of the Lys3–Ala7segment. Inter-
residues dipolar interactions observed in the ROESY experiment
between H-? of Lys3and H-? of Pro4indicate that Pro4adopts a trans-
conformation in the major conformer of p100c. In addition to standard
sequential interactions, several medium range interactions were also
observed between side chain protons of residues Pro4to Ala7as for
example between all protons of Pro4and the methyl group of Ala7(Fig.
1). Moreover, four long range ROE connectivities were observed
between Tyr2and His10protons, confirming the cyclic nature of the
peptide (Fig. 1). ROE derived distances and coupling constants were
used as constraints to generate a family of structures for p100c using
DYANA.The10beststructuresindicatethatthePro4–Pro7fragmentof
p100c is conformationally organized into a type I ?-turn (Fig. 2 and
Table 2) (61, 62). Because both the cyclic form and Pro4can induce this
typeofconformationalbehavior,thestructuralanalysiswasextendedto
reduced p100c (data not shown). The type I ?-turn remained, suggest-
ing that Pro4alone is responsible for its formation, although the cyclic
structure might contribute to its stabilization.
Peptide22(KRHFLSQRQ)—Availabledata(Table4)suggestthatp22
is very flexible. Indeed, except for the slight deviation observed for the
Ser6H-?proton,chemicalshiftsdonotsignificantlydeviatefromstand-
ard values. Meanwhile, none of the coupling constants of internal resi-
dues could be measured because of extensive signals overlaps. Never-
theless, medium range ROE connectivities were observed between side
chains protons of residues His3and Leu5as well as between those of
Phe4and Ser6(Fig. 1). The 10 structures of lowest energy matching
those distance constraints show that fragment His3to Ser6of p22 is
organizedintoanonclassified?-turn(Table2),althoughthepeptideN-
andC-terminalendsremainquiteflexible(Fig.2).Thatavailablechem-
ical shift and coupling constant values do not reflect such an organized
conformation in solution suggests a weaker stability of the ?-turn.
Ligand Interaction with the Protective mIgAs—Investigation of the
molecular pattern of the interactions involved in the peptide- and pen-
tasaccharide-mIgA complexes relied on two complementary method-
ologies, namely trNOE and STD NMR experiments, whose combina-
tion was found to model accurately mAb-ligand interactions (38, 47).
Indeed, the former technique provides key information on the confor-
mation of the bound ligand, whereas the latter allows epitope mapping
viamagnetizationtransferfromtheproteintotheresiduesoftheligand
that are in close contact with the protein. To carry out these experi-
ments a few requirements have to be fulfilled as follows: (i) have an
importantcontributionfromtheboundstatetotheNOEs,and(ii)have
an exchange rate that is fast enough compared with the free ligand
longitudinal relaxation. Because here the IC50was the sole information
available in terms of binding parameters for the complexes, for each
system the best ligand:mIgA ratio was first evaluated from titration
experiments according to the method of try and assay.
Binding of DA(E)BC-OMe to mIgA I3 and mIgA C5—Bound pen-
tasaccharide conformation. Independently of the mIgA tested, the best
TABLE2
Characterization of ?-turn types of peptides p115, p100c, and p22 free and bound to protective mIgAs
Position i ? 1
? (°)
PeptidesTurnsa
Position i ? 2
?-turn types
? (°)
? (°)
? (°)
Ramachandran
nomenclatureb
?p?
??
?p?
??
??or??
??
?E?
Classical
nomenclaturec
p115 freePPWA
PWAR
PPWA
PLGA
PLGA
HFLS
HFLS
?75.0 ? 0.1
?50.7 ? 0.4
?75.0 ? 0.1
?88.8 ? 27.2
?95.0 ? 7.2
159.8 ? 5.8
?121.6 ? 2.3
?32.8 ? 0.2
?15.0 ? 0.3
140.0 ? 0.4
?66.6 ? 19.0
64.2 ? 23.0
?30.3 ? 19.4
122.5 ? 0.8
?50.7 ? 0.4
?46.6 ? 0.1
97.5 ? 0.1
?93.3 ? 27.7
163.9 ? 23.1
-153.3 ? 21.1
55.7 ? 0.1
?15.0 ? 0.3
-20.4 ? 0.2
20.7 ? 0.1
6.1 ? 12.1
?41.3 ? 13.0
?18.3 ? 13.1
76.7 ? 0.5
I
p115 mIgA C5
p100c free
p100c mIgA I3
p22 free
p22 mIgA I3
aResidues in turn conformation.
b?-Turn types as defined by Wilmot and Thornton (91).
cRichardson classification system (92).
II
I
?II
?II
FIGURE2.Structuresofthemajorconformersofpeptidesintheirfree(A)andmIgA-
bound (B) forms. p115 (on top), p100c (in the middle), and p22 (in the bottom) are
shown. For each peptide, the backbone of the 10 best calculated structures are super-
imposedovertheresiduesin?-turnconformation.B,thepeptidessidechainsincontact
with mIgAs, according to STD experiments, are shown in green.
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DA(E)BC-OMe:mIgA ratio to observe trNOEs was shown to be 20:1 in
binding sites. Because the highest attainable mIgA concentration was
3.75 ?M, a 0.3 mM concentration of DA(E)BC-OMe was used to fulfill
the 20:1 ratio requirement. Because NOE intensities depend, among
other parameters, on the correlation time for reorientation and there-
fore on temperature, the later parameter was optimized so that ??c
equals 1 for the free pentasaccharide, thus allowing us to distinguish
NOE from trNOE connectivities. Indeed, the NOESY spectrum of the
freepentasaccharideat30 °CdisplayedonlyfewpositiveandweakNOE
connectivities characteristic of small molecules, whereas negative NOE
connectivities were observed in the trNOESY spectrum of DA(E)BC-
OMe when interacting with either mIgA C5 or mIgA I3. Because these
effects did not result from the increased solution viscosity as probed by
recordingNOESYspectraofthepentasaccharideinthepresenceofBSA
at the same w/v concentration (37), it was assumed that they corre-
sponded to trNOE connectivities for the mIgA-bound DA(E)BC-OMe.
trNOESY spectra obtained with several mixing times, ranging from
100 to 400 ms, allowed us to trace the build-up curves (trNOEs inten-
sities versus ?m) from which the distance information was extracted. In
addition, inter-residue1H–1H distances were also calculated from a
trROESY spectrum obtained with a mixing time of 400 ms to take spin
diffusionintoaccount,ifany.Comparisonofthesedistanceswiththose
measured for unbound DA(E)BC-OMe (33) suggested that the pen-
tasaccharideconformationwasnotsignificantlymodifieduponbinding
to the mIgAs (Table 5).
Epitope Characterization—The key elements involved in DA(E)BC-
OMe binding to the mIgAs were then characterized based on STD
experiments.AsforthetrNOEexperiments,anoligosaccharidetomAb
ratioof20:1inbindingsitewasused.Todecreasetheexchangerate,the
temperature was set at 15 °C. The one-dimensional STD spectrum of
DA(E)BC-OMe interacting with mIgA C5 shows that protons H1 and
H2 and H6 (methyl group) of rhamnoses A and B, as well as all protons
belongingtoglucose(E),areinclosecontactwiththemIgAC5-binding
site(Fig.3).Indeed,theseinteractingprotonswerefullyidentifiedinthe
corresponding two-dimensional STD-TOCSY spectrum (Fig. 4). Simi-
lar results were obtained for DA(E)BC-OMe binding to the mIgA I3
(data not shown). Furthermore, protons from the glucose (E) and the
methyl group of residue B give the strongest signal enhancements, sug-
gestingthattheyareinclosestcontactwithbothmIgAs(Fig.5)andplay
a crucial role in the oligosaccharide-mAb interaction.
Binding of the Peptide Mimics to mIgAs—Based on available IC50
values, analysis was run on the p115-mIgA C5, p100c-mIgA I3, p22-
mIgA C5, and p22-mIgA I3 complexes.
Interaction of Peptide 115 (KVPPWARTA) with mIgA C5—trNOE
experiments recorded for the p115-mIgA C5 complex (see supplemen-
talFig.4S)showednewNOEconnectivitieswhencomparedwiththose
observed for the free peptide. These additional cross-peaks, such as
those observed between residues Trp5and Ala6, were clearly identified
as representative of the p115-bound form. Interestingly, most of the
NOE connectivities involving amide protons of the free p115 were no
longer observed in bound p115 with the exception of the Trp5, Ala6
amide proton connectivity (see supplemental Fig. 1S). The pH increase
from 5 in the free peptide to 6.5 in the peptide:mIgA solution might
account for such experimental observations since amide protons
exchange faster at higher pH. Nevertheless, the medium range NOE
connectivities observed between the side chain proton of residues Val2
and Pro3, and the CH3-? of Ala6remained (see supplemental Fig. 1S).
DistanceconstraintsderivedfromtrNOEintensitieswereusedtoestab-
lish the conformation of p115 when bound to mIgA C5. Superimposi-
tion of the 10 lowest energy backbone conformations of free p115 to
thoseofmAb-boundp115showedthatonlytheturninvolvingresidues
Pro3toAla6,observedinthefreeform,ismaintainedintheboundform.
Based on C-?i–C-?i?3distances as well as on ? and ? angle values
(Table2)thetypeI?-turnobservedbetweenresiduesPro4andArg7for
thefreepeptideisnolongerpresent.Whereasinthefreeformfragment
TABLE3
1H chemical shifts of the trans-isomer of p100c in H2O/D2O (90/10),
pH 5.1 and 298 K
Chemical shifts measured in ppm with an accuracy of ?0.01 ppm are referenced to
external 4,4-dimethyl-4-silapentane sodium sulfonate (?H0.00).
Residue
Cys1
ND
Tyr2
4.65
?0,10c
Lys3
8.05 (8.00)4.47
?0,13
HN
NDa
H?
H?
Others
3.29–3.22
3.04–2.91 8.87 (7.20)b
H-? 7.12
H-? 6.80
H-? 1.23
H-? 1.63
H-? 2.90
H-? 7.45
H-? 1.88
H-? 3.39
H-? 1.61
H-? 0.92–0.88
1.55
Pro4
4.30
?0,12
4.23
?0,11
4.05–3.74
?0,09/?0.22
4.31
?0,01
4.37
0,03
4.30
?0,05
4.38
?0,35
4.51
?0,20
2.23–1.84
Leu5
8.22 (5.30)1.61
Gly6
8.46 (5.40)
Ala7
8.02 (5.40)1.38
Leu8
8.32 (7.20)1.69 H-? 1.62
H-? 0.92–0.85
H-? 1.15
Thr9
7.69 (7.80) 4.17
His10
8.30 (7.80) 3.36H-?1 8.58
H-?2 7.31
Cys11
8.49 (7.40)3.28–3.05
aND indicates not determined.
bData in parentheses are3JHN,H?coupling constants (in Hz ? 0.2 Hz) measured
from the one-dimensional spectrum.
cData in italics are the difference between the H? chemical shifts of the residues of
thepeptideandthoseofthesameresiduesinnonstructuredpeptidesGGXAGGor
GGXPGG (90).
TABLE4
1H chemical shifts of the trans-isomer of p22 in H2O/D2O (90/10), pH
5.1 and 298 K
Chemical shifts measured in ppm with an accuracy of ?0.01 ppm are referenced to
external 4,4-dimethyl-4-silapentane sodium sulfonate (?H0.00).
Residue
Lys1
3.88
?0,41
HN
NDa
H?
H?
1.80
Others
H-? 1.36
H-? 1.67
H-? 2.96
H-? ND
H-? 1.52
H-? 3.14
H-? ND
H-?1 7.86
H-?2 6.98
H-? 7.20
H-? 7.29
H-? 7.33
H-? 1.50
H-? 0.89–0.84
Arg2
ND4.28
?0,06
1.68
His3
ND4.76
?0,03
4.59
?0,03
3.18–3.00
Phe4
8.22 (7.20)3.09–2.97
Leu5
8.33 (ND)4.32
?0,02
4.38
?0,09
4.35
0,01
4.32
?0,02
1.57
Ser6
8.22 (ND) (6.70) 3.85
Gln7
8.35 (ND)2.11–1.96 H-? 2.34
H-? 6.82–7.53
H-? 1.62
H-? 3.18
H-? ND
H-? 2.28
H-? 6.78–7.50
Arg8
8.33 (ND)1.86–1.74
Gln9
8.04 (7.70)4.15
?0,19
2.09–1.90
aND indicates not determined.
bData in italics are the difference between the H? chemical shifts of the residues of
thepeptideandthoseofthesameresiduesinnonstructuredpeptidesGGXAGGor
GGXPGG (90).
cData in parentheses are3JHN,H?coupling constants (in Hz ? 0.2 Hz) measured
from the one-dimensional spectrum.
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Pro3–Ala6adopts a nonclassified type of ?-turn, in the bound form a
welldefinedtypeII?-turnisclearlypresent(61,62)(Fig.2andTable2).
STD experiments (Fig. 6), run under the experimental conditions
used for trNOE experiments, showed that the p115 protons in close
contactwithmIgAC5arethesidechainprotonsofLys1andPro4,allthe
methyl groups thus implicating Val2, Ala6, and Ala9, and all protons of
theTrp5aromaticring.Clearly,inadditiontosideinteractionsinvolving
the N- and C-terminal ends, major contacts involve residues from the
Pro4– Ala6segment, suggesting that the structured Pro3–Ala6type II
?-turn is crucial for p115:mIgA C5 recognition. It is worth noting that
the major form of p115 was that recognized by mIgA C5.
Interaction of Peptide p100c (CYKPLGALTHC) with mIgA I3—As
comparedwithdatacorrespondingtothefreeform,thetrNOESYspec-
trum of p100c in interaction with mIgA I3 displayed new data specific
for the bound form. These include new sequential NOEs such as those
observed between Lys3and Pro4or between Pro4and Leu5, whereas
sequential NOEs between Ala7and Leu8, or between Leu8and Thr9,
were no longer visible. However, medium range NOE connectivities
FIGURE3.mAbbindingepitopeofthepentasaccharideDA(E)BC-OMe.A,one-dimensional1HreferencespectrumofpentasaccharideDA(E)BC-OMeinthepresenceofmIgAC5
(20:1 ratio in site). B, one-dimensional STD-NMR of DA(E)BC-OMe in the presence of mIgA C5 with selective saturation of antibody resonances at 0.3 ppm. Protons of DA(E)BC-OMe
affected by the selective saturation of mIgA C5 and so in contact with the mAb are labeled.
TABLE5
1H-1H inter-residue distances (Å) extracted from dipolar interactions observed in ROESY and NOESY spectra of DA(E)BC-OMe pentasaccharide
free and in interaction with mIgA C5 and mIgA I3
The two values correspond to distances extracted from ROESY (400 ms) (left) and NOESY (right), respectively (accuracy, ?10%). ND indicates not determined.
Proton pairsa
A-1/B-13.3/ND
A-1/B-22.2/ND
A-5/B-12.4/2.5
A-6/B-13.4/3.4
B-1/C-32.3/2.3
B-2/E-1 2.3/ND
B-3/E-1 2.5/2.5
B-3/E-53.3/3.2
B-6/C-2 3.6/3.5
aA-1 corresponds to proton 1 of rhamnose A.
DA(E)BC-OMeDA(E)BC-OMe/IgA C5
ND/3.0
ND/2.4
ND/2.7
ND/3.2
ND/2.3
ND/2.5
ND/3.2
ND/2.7
ND
DA(E)BC-OMe/IgA I3
ND/2.9
2.1/2.2
2.4/2.5
ND/3.2
2.3/2.2
2.2/2.3
2.8/2.8
ND/2.7
ND/3.5
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observed between Pro4and Ala7in the free form remained. In addition,
medium range interactions specific to the bound form were observed,
such as those involving side chain protons of Tyr2and Leu5, Lys3and
Leu8, as well as Tyr2and His10(see supplemental Fig. 2S). Superimpo-
sition of the backbone (Pro4to Ala7fragment) of the 10 lowest energy
conformationsoffreeandmIgAI3-boundp100cmatchingthedistance
constraints showed that, as observed for p115, the turn observed in the
freeformwasmaintainedintheboundform.Furthermore,datapointed
toaswitchfromatypeI?-turninthefreeformtoatypeII?-turninthe
bound form (Table 2) (61, 62). Additional significant rearrangements
were observed for the rest of the backbone (Fig. 2).
More detailed epitope identification was derived from the one-di-
mensional STD experiment. All methyl group protons of p100c, thus
involving residues Leu5, Ala7, and Leu8as well as the Tyr2side chain
FIGURE 5. Structure of the pentasaccharide DA(E)BC-OMe. The representation of a
lowest energy conformation of DA(E)BC-OMe as determined with the CICADA method
andinagreementwithNMRdata(33).Thesmallspheresindicatetheprotonsthatarein
contact with the protective mAbs, mIgA C5, and mIgA I3, in agreement with the STD-
NMR experiments.
FIGURE4.ThebranchedglucoseresidueEofthe
pentasaccharide DA(E)BC-OMe constitutes a
key element in mAb recognition. A, two-dimen-
sional STD-TOCSY of DA(E)BC-OMe in the pres-
ence of mIgA C5 (20:1 ratio in site) with selective
saturation of antibody resonances at 0.3 ppm. B, a
zooming of the two-dimensional STD-TOCSY in
the 3.4–4 ppm region emphasizing the glucose E
proton connectivities.
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protons and the Lys3CH2-? protons, were in contact with the mIgA I3
(Fig. 7). Within the turn, only Leu5and Ala7methyl groups contacted
the mAb. Similarly to p115, the methyl groups of the hydrophobic res-
iduesandthearomaticresidueofp100cwereinvolvedintheinteraction
with the mIgA I3.
InteractionofPeptidep22(KRHFLSQRQ)withmIgAI3andmIgAC5—
Comparison with the spectrum of the free peptide shows that the trNOE
connectivities observed when p22 is bound to mIgA I3 differ for medium
rangeinteractions.Indeed,dipolarinteractionsobservedbetweenPhe4and
Ser6infreep22disappearedtothebenefitofnewinteractionsbetweenHis3
and Ser6in the bound form. However, dipolar interactions between His3
andLeu5wereobservedbothinthefreeandtheboundforms(seesupple-
mental Fig. 3S). Comparison of the lowest energy conformations adopted
by p22 in the free and bound forms showed that the nonclassified ?-turn
involving the His3–Ser6segment in the free form changed to a type II
?-turn in the bound form, as observed for p115 and p100c. Additional
rearrangementswereobservedfortherestofthebackbone(Fig.2).trNOE
experiments for p22 bound to the mIgA C5 were unsuccessful because
sparsenegativeNOEswereobserved(datanotshown).Thehighaffinityof
mIgA C5 (IC50of 0.03 ?M), most probably associated with an equilibrium
constant for dissociation (Kd) below 10?6M and thus not compatible with
trNOE observations (10?3? Kd? 10?6M), might be responsible for this
effect. Despite this drawback, epitope mapping by STD experiments was
successfullyundertakenasthelowerlimitforexchangewaslessstringentin
termsofKd(10?8M).Epitopeidentificationforp22boundtomIgAI3and
mIgAC5,respectively,wasobtainedfromtheone-dimensionalSTDexper-
iments(Fig.8).WhethermIgAI3orIgAC5wasconcerned,p22residuesin
directcontactwiththemAbswereidentical.TheyincludedtheHis3imid-
FIGURE 6. mAb binding epitope of the peptide p115. A, one-dimensional reference spectrum of p115 in the presence of mIgA C5 (20:1 ratio in binding site). B, one-dimensional
STD-NMR of p115 in presence of mIgA C5 with selective saturation of mAb at 0.3 ppm. Protons of p115 affected by the selective saturation and so in interaction with mIgA C5 are
labeled.
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azole protons, Phe4aromatic protons, as well as Leu5methyl protons, all
corresponding to amino acids taking part in the turn observed in free p22.
AlthoughtheconformationofmIgAC5-boundp22remainedundisclosed,
available data suggest that the nonclassified turn naturally adopted by p22
contributedtobothmIgAI3andmIgAC5recognition.
Modeling of the Fab Domain of mIgA I3—AblastsearchintheProtein
DataBank(55)allowedustoidentifythreemAbchainswithhighsequence
similaritytoeithertheVLortheVHchainofmIgAI3.Sequencealignments
are displayed in Fig. 9 together with Protein Data Bank code for the struc-
tures of interest that include anti-RNA mAb (code 1MRD) (63), the anti-
DNA mAb (code 1CBV) (64), the catalytic mAb (code 1A4J) (65), anti-
influenza neuraminidase (code 1NCA) (66), and anti-cholera toxin mAb
(code 1TET) (67). Each chain was built by homology modeling using the
standard procedure of the composer program (56). The murine Fab frag-
mentwithDiels-Aldercatalyticproperties(65)displayedhighsimilarityfor
bothchainsandwasusedasatemplateforassemblingthetwochains.Asa
generalfeatureformAbs,theH3loopofCRDisknownasthemostvariable
one. Among the structures with high homology, the H3 loop of the anti-
cholera toxin Fab (67) was selected as a template because it displays the
same number of amino acids as the target. After optimization of the side
chainconformation,thebindingsiteofmIgAI3appearedtohaveadistinct
“groove” character located between the variable loops with a deep central
pocket. The sides of the groove were flanked by the CDRs, mostly H2 and
H3oftheheavychainandL1inthelightchain.
FIGURE 7. mAb binding epitope of the peptide p100c. A, one-dimensional reference spectrum of p100c in presence of mIgA I3 (20:1 ratio in binding site). B, one-dimensional
STD-NMR of p100c in presence of mIgA I3 with selective saturation of mAb resonances at 0.3 ppm. Protons of p100c affected by the selective saturation and so in interaction with
mIgA I3 are labeled.
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DockingofOligosaccharidesandModelingofComplexeswithmIgAI3—
PreviousstudiesidentifiedtwodifferenthelicalconformationsofS.flexneri
5a O-SP as being the most stable ones in solution (33). Both can be
describedasright-handed3-foldhelices,butoneismoreextended(E)than
the other (O) with helical repeats of 23.2 and 19.4 Å, respectively. The
nonasaccharide BCDA(E)BCDA was selected for docking studies as the
largest O-SP fragment that can be treated as a flexible ligand. When the
nonasaccharide BCDA(E)BCDA fixed in both conformations is docked
into the Fab-binding site, four solutions can be identified, two for the E
conformationandtwofortheOone.Inbothcases,thenonasaccharidecan
fit either with a parallel orientation to the binding site groove or with a
perpendicularone.Inanycase,thebranched?-D-glucopyranoseresidueE
is deeply buried into the central pocket of the groove. In all of the four
solutions, the mAb features involved in carbohydrate recognition are the
three loops from the heavy chain and the L1 and L3 loops from the light
chain.ThevariableloopH3playsthemajorrole,withitstwoAspresidues
(Asp91andAsp92)involvedinrecognitionformostbindingmodes.
When the nonasaccharide-bound conformations were propagated
into polysaccharide structures comprising four repeating units, only
two docking modes appeared to be stable with additional contacts cre-
FIGURE8.mAbbindingepitopeofthepeptidep22.A,one-dimensionalreferencespectrumofp22inpresenceofmIgAI5(20:1ratioinbindingsite).B,one-dimensionalSTD-NMR
ofp22inpresenceofmIgAI3.C,one-dimensionalSTD-NMRofp22inpresenceofmIgAC5.TheselectivesaturationofmAbresonanceswasdoneat0.3ppm.Protonsofp22affected
by the selective saturation and so in contact with the mAbs are labeled.
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ated on both sides of the binding site. In each case, this ability corre-
sponded to the parallel arrangement of BCDA(E)BCDA. Therefore,
only these two solutions, i.e. docking of E and O conformations in par-
allelmode,wereconsideredaspossiblemimicsofO-SPbindingtomIgA
I3.ThetwopossibledockingmodesofBCDA(E)BCDAandthepolysac-
charideofDP4aredisplayedinFig.10(A–D)andthecontactsofinterest
are listed in Table 6. For both conformations of each ligand, the central
trisaccharide A(E)B makes most of the binding contribution, and the
additional contacts established by the GlcNAc (D) residue are minor.
Docking of Peptide Mimics—Docking of peptides to mAbs was per-
formedononeexampleinordertorationalizetheprotectiveeffect.The
cyclic peptide p100c is conformationally constrained and was therefore
selectedforthemodelingstudieswiththemAbthatdisplaysthehighest
affinity, i.e. mIgA I3. For the two lowest energy docking solutions, the
p100C conformation displayed good shape complementarity with the
binding site central pocket of mIgA I3. In the first solution, a strong
interaction (three H-bonds and two salt bridges) appeared between the
peptide and the mAb, mainly located on CDRH3 domain involving
Asp91andAsp92(Table6).Theseconddockingsolutionledtoidentical
main interactions between p100c and CDRH3 (Table 6). In both cases,
the Leu5–Gly6–Ala7motif of p100c, seen as constrained by NMR, was
driven into the central pocket (van der Waals interactions). Both dock-
ing modes allowed for a strong interaction between the Lys3of the
peptide and the protein Asp92. Nevertheless, the Tyr2residue of the
peptidewasburiedinthesecondsolutionandestablishedstrongvander
WaalscontactswiththearomaticsidechainofTrp41andPhe94ofmIgA
I3. Therefore, this model displayed in Fig. 10 (E and F) was that in best
agreement with NMR data.
DISCUSSION
Asanovelstrategytoimprovevaccinedesign,molecularmimicryhas
gainedagrowinginterestintherecentpast.Formimicryofpolysaccha-
rides, the mimics can be of the same molecular class as the natural
antigen, i.e. oligosaccharides, or different, as for instance the peptide
mimics. The nature of peptide carbohydrate mimicry has not yet been
deciphered, and detailed structural studies of both oligosaccharide-
mAb complexes and carbohydrate-mimicking peptides-mAb com-
plexesarestillneededtoexpandthethusfarlimitedstructuraldatabase
available in the series. Peptide-carbohydrate mimicry is either struc-
tural, functional, or both. Structural mimicry resides in mimicry of spe-
cific chemical groups of the carbohydrate by chemical groups of the
peptide, thus both ligands contact the same residues in the mAb-bind-
ingsite(68).Mimicryistermedfunctionalwhenthemimicdiffersstruc-
turally from the natural antigen. Both the antigen and the mimic cross-
react specifically with the mAb used for selection, although the protein
residues involved in recognition differ (17, 21, 69).
Here, by aiming at designing new vaccine strategies against Shigella
infection, we developed synthetic mimics, carbohydrates and peptides,
of S. flexneri 5a O-SP. Previous NMR and molecular modeling studies
from our laboratory have shown that, among the four possible frame-
shifted pentasaccharides representative of the O-SP, DA(E)BC-OMe
best mimics the conformational features of S. flexneri 5a O-SP (33).
More importantly, the trNOE data reported here indicate that the con-
formationofDA(E)BC-OMewhenboundtoO-SP-specificmIgAI3and
mIg C5 does not differ from its conformation when free in solution.
Noteworthy, selection of free solution conformers often predominates
in mAb-carbohydrate recognition processes (70). However, it is not
always so as exemplified with mAb Se155-4 binding to a trisaccharide
antigenic determinant of the Salmonella paratyphi B branched O-SP
(71).
Anotherinterestingexampleofsuchinducedconformationalchange
isthemAbSYA/J6bindingtothepentasaccharideABCDA?fragmentof
the linear O-SP defining S. flexneri serotype Y (72). Modeling of the
linear heteropolysaccharide has shown that it is structured into a left-
handled helical chain of three ABCD repeating units (73, 74). Most
interestingly, extension of the modeling study to S. flexneri 5a O-SP
FIGURE 9. Alignment of mIgA I3 sequence with
relatedsequencestakenfromtheProteinData
Bank. Sequence differences are highlighted by
displaying amino acid code using white letter on
blackbackground.
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suggested that residue E, which is associated with serotype specificity,
crucially impacts the overall O-SP conformation. In fact, the branched
heteropolysaccharide, whose repeating unit (I) bears the E side chains,
behaves as a right-handed 3-fold helix with residue E protruding out-
wardly (33). Along this line, the interaction of DA(E)BC-OMe with the
serotype-specific mIgAs is mainly driven by the branched E residue as
evidenced by the large number of E-specific signals enhanced in the
STD spectra of the pentasaccharide in complex with mIgA I3. Further-
more, NMR experiments showed that all rhamnose methyl groups are
also in close contact with the mAb, with the methyl group of rhamnose
B on which E is branched being the major contributor. The N-acetyl
group of residue D gives only weak contacts with the mIgA protons,
indicatingthatitprobablyliesatthesurfaceofthemAbs.Thesedataare
supportedbytheinhibitionELISAresultsshowingthatallframe-shifted
tri-,tetra-,andpentasaccharides,bearingA(E)B-branchedtrisaccharide
characteristic of S. flexneri 5a serotype, are recognized by a protective
serotype 5a-specific mIgG (33). Further insights on the central role
played by the branched ?-D-glucopyranosyl residue E in mAb recogni-
tion derives from docking of the nonasaccharide BCDA(E)BCDA and
fragments of the O-SP comprising four repeating units in the mIgA I3
Fab-binding site. The latter appears to have a distinct groove character
with a deep central pocket, a type of binding site often encountered for
mAbs binding internal polysaccharide sequences (75). Most interest-
ingly,thisfindingisidenticaltothatobservedforStrep9,amousemAb
of the IgG3 subclass directed against the cell wall polysaccharide of
group A Streptococcus (76) made of repeats comprising a branched
?-N-acetyl-D-glucosamine residue linked to a linear di-rhamnopyrano-
syl backbone. As found earlier, sides of the mIgA I3 groove are flanked
by CDR2 of the heavy chain and CDR1 of the light chain. Moreover,
aromaticresiduessuchasTyr45oftheheavychainandTyr34ofthelight
chaindefinethepocketregion,pointingoncemoretotheimportanceof
suchaminoacidsincarbohydraterecognition(77,78).Indeed,whatever
the orientation of nonasaccharide BCDA(E)BCDA, parallel or perpen-
dicular relative to the groove binding site, the glucose residue E was
always deeply buried in the central pocket of the groove and was poorly
accessibletosolvent.Mostinterestingly,relyingonmolecularmodeling
only, a heptasaccharide related to Brucella abortus O-SP exemplifies
another O-SP-mAb interaction for which the mAb-binding site identi-
fied as a groove bearing a pocket in its center could also accommodate
twobindingmodesofanO-SPfragment(79).Asshownhere,dockingof
S. flexneri 5a O-SP large fragments in the mIgA I3-binding site pointed
to only one possible binding mode of the O-SP, namely the parallel
mode, independently of the length of the helical repeat taken into
account. Thus, in addition to the branched glucopyranosyl residue E
behaving as an anchor and to the trisaccharide A(E)B providing the
criticalepitopeexposedontheO-SP,chainelongationalsotakespartin
O-SP:mIgArecognition,highlightingtheessentialcontributionofsome
kind of conformational epitope or presentation in an extended surface.
However, we are aware that small changes at the VL:VHinterface of the
mAb may result in significant alteration of the binding mode, which
cannotbepredictedatthisstage(80).Thus,dataprovidedhereareonly
meanttoprovideamodelofS.flexneri5aO-SPbindingtoahomologous
protective mIgA, which needs to be further assessed based on crystal-
lographic data.
FIGURE 10. Graphical representation of the different models of antibody mIgA I3
(light chain in green and heavy chain in violet) with docked oligosaccharide and
cyclicpeptidep100c.AandB,twopossibledockingmodesfornonasaccharideinmIgA
I3. C and D, corresponding interaction with the polysaccharide (four repeating units)
afterpropagationofthenonasaccharideconformationalongthe20-residuechain.Eand
F, two different views of the docking mode of p100c in mIgA I3-binding site displaying
the best agreement with NMR data.
TABLE6
Contact between ligand and antibody in the different model for
docking oligosaccharide and cyclic peptide p100c
Ligand atom
Oligosaccharide model 1 (extended)
D:GlcNAc.O4-H
D:GlcNAc.O6-H
D:GlcNAc.O6
A:Rha.O3-H
A:Rha.O4
E:Glc.O3-H
E:Glc.O3
B:Rha.O4-H
Oligosaccharide model 2 (extended)
D:GlcNAc.O6
E:Glc.O2-H
E:Glc.O3-H
E:Glc.O3
B:Rha.O4-H
A:RhaO4
Peptide p100c model 1
H-bonds
Lys3.NH3?
Lys3.NH3?
Leu5.O
His10.NH?
His10.NH?
Hydrophobic interactions
Pro4
Ala7
Peptide p100c model 2
H-bonds
Tyr2.NH
Tyr2.OH
Lys3.NH3?
Hydrophobic interactions
Tyr2
Pro4
Pro4
Pro4
Protein atomA? chain
Ile22.O
Ala90.O
Gly24.NH
Asp92.COO?
Lys30.NH3?
Asp91.COO?
Tyr93.NH
Ser93.O
H1
H3
H1
H3
L1
H3
H3
L3
Gly24.NH
Asp91.O
Asp91.O
Tyr93.NH
Ser93.O
Ser29.OH
H1
H3
H3
H3
L3
L1
Asp91.COO?
Asp92.COO?
Gly24.NH
Asp28.COO?
Ser29.OH
H3
H3
H1
L3
L3
Trp41
Trp41
L2
L2
Ile22.O
Asn26.NH2
Asp92.COO?
H1
H1
H3
Trp41
Trp41
Phe94
Pro98
L2
L2
H3
H3
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Peptides cross-reacting with S. flexneri 5a O-SP have been identified
byscreeningphage-displayedpeptidelibrarieswithprotectivemIgAC5
and mIgA I3 (28). Nonconstrained and constrained peptide libraries
were screened. Indeed, it is expected that constraining a peptide limits
its flexibility and therefore may improve its affinity for mAb binding,
and consequently, may allow the selection of better mimics of the nat-
ural antigen (81). All selected peptides, whether mimotopes or mimics
only,exhibitedanIC50valuerangingfrommicromolartosubmicromo-
lar (this work and Footnote 5). They were better recognized than the
pentasaccharide best mimicking S. flexneri 5a O-Ag (IC50in the milli-
molarrange)byatleastoneofthemIgAsusedforselection.However,as
outlined previously for other systems (17, 82, 83) and observed here for
p115 and p100c, but not for p22, most selected peptides could discrim-
inate between the two mIgAs used for selection. This is in agreement
withtheassumptionthatanti-polysaccharidemAbsmaynotnecessarily
recognizeasingleantigentopography.Inlinewithpreviouswork(83),it
was thus hypothesized that peptide mimics reacting with a panel of
anti-O-SP-specific mAbs would have a better potential to act as mimo-
topes than those with a strong discriminating potential. However, as
observedbyothers(18),ourdatadonotsupportthishypothesis.Indeed,
among the three sequences selected for the study, discriminating p115
and p100c behave as mimotopes, whereas p22, recognized by both
mIgAs, is only a mimic of S. flexneri 5a O-SP. Besides, considering the
highaffinityofp22formIgAC5(IC50?30nM),ourmodelfitstoothers,
suchasthatonCryptococcusneoformans(18,84)andthatonN.menin-
gococcus C (30), suggesting that commonly used parameters for select-
ing peptide mimicking polysaccharide antigens, such as high-affinity
bindingtomAb,arenotpredictiveoftheabilityoftheselectedpeptides
to act as mimotopes. Indeed, based on x-ray analysis, several lines of
evidence support the idea that peptide binding to an anti-polysaccha-
ride mAb may differ significantly from that of the natural antigen. On
one hand, data on the dodecapeptide PA1 mimicking C. neoformans
CPS suggest poor steric complementarity between PA1 and the heavy
chain of the mAb used for selection, which may explain why PA1 acts
only as a partial mimotope (20). On the other hand, an octapeptide
functional mimic of S. flexneri serotype Y O-SP was found to comple-
menttheshapeofthegroove-typebindingsiteofmAbSYA/J6,usedfor
selection, much better than the ABCDA? pentasaccharide fragment of
the O-SP (21). However, the peptide does not fully complement the
deep pocket located in the center of the groove and occupied by rham-
nose C upon binding of ABCDA?. This may explain, at least in part, the
poor ability of the octapeptide to behave as an immunogenic mimic (9).
Not surprisingly, although the three peptides do not share any con-
sensus sequence, the conformations they adopt in their free form
encompass many rapidly interconverting conformers with short inter-
nal sequences spending long lifetimes organized in turn like motifs.
Although p22 was found much more flexible than p115 or p100c, all
three peptides adopted ?-turn conformations, either of nonclassified
type or of type I. This appears to be a rather common conformational
featureforshortpeptidesrepresentativeofantigenicregionsofproteins
(85) or polysaccharide antigens such as group A Streptococcus CP (9)
and group B Streptococcus CP (38). Indeed, it has been suggested that a
?-turnallowsappropriateexposureofsidechainresiduesforoptimalfit
withinthemAbcombiningsite.Mostinterestingly,inthelaterexample,
peptide FDTGAFDPDWPA, a molecular mimotope of the CPS, was
earlier thought to adopt a nonrandom coil conformation in aqueous
solution assimilated to a nascent helix that could potentially mimic the
extended helical form of the natural carbohydrate epitope (29). This
discrepancyunderscoresthehighcomplexityofconformationalstudies
dealing with short peptides. The relative heterogeneity of ?-turn types
adopted by p115, p100c, and p22 in the free form is completely lost in
the bound form, as the three peptides adopt a type II ?-turn conforma-
tion, which appears to be crucial for binding independently of the
involvedmAb.Thus,thelackofconsensussequenceamongtheselected
peptides seems to be compensated by structural consensus induced
upon fitting to the mAb combining sites. Moreover, the type II ?-turn
structure starts from a proline (Pro3and Pro4, respectively) and ends
with an alanine (Ala6and Ala7, respectively) for both p115 and p100c,
underscoring the partial structural resemblance between the two pep-
tides.Majorcontributionsofthep115-turntobindinginvolvearomatic
Trp5and cyclic Pro4, whereas hydrophobic Leu5was the residue most
involved in mAb binding to the p100c-turn. p22 differs notably from
p115andp100cbecauseithasnoproline.Inthiscase,aromaticHis3and
Phe4together with hydrophobic Leu5are the major turn components
contributing to binding independently of the mAb. Noteworthy, addi-
tional residues do not seem to be engaged in mAb recognition, which
may explain the ability of p22 to bind the two mIgAs. On the contrary,
goingfromHis1toAla9,p115bindingtomIgAC5necessitatesthatmost
residues along the peptide chain, especially those at the N terminus,
makespecificcontactswiththecombiningsite.Similarly,residuesatthe
N terminus of p100c appear critical for peptide binding to mIgA I3. In
particular, the two docking models obtained for p100c interacting with
mIgA I3 reveal a salt bridge formed between the peptide Lys3residue
and the residue Asp92within the CDR H3 loop, similarly to those
observed between rhamnose A or glucose E and Asp92or Asp91of the
mIgACDRH3loop,respectively,uponDA(E)BCbinding.Althoughnot
probed at this stage, analogous ionic contributions to peptide-mAb
interactionsmaybeanticipatedbecauseallselectedpeptidessharebasic
residues.However,availabledatasuggestthatindependentlyofthepep-
tide mimic under study, all mIgA-peptide interactions derived mostly
fromthedirectcontactofpeptidearomaticresiduesandmethylgroups
with the mAb-binding site, suggesting that recognition was basically
driven by hydrophobic and van der Waals contacts. In that matter, data
provided for the S. flexneri 5a system fully support previous observa-
tions made for other models implicating peptides mimicking polysac-
charide antigens in complex with specific mAbs (11).
In addition, all data reported here strongly emphasize the crucial role
playedbythetypeII?-turntopologyingoverningmolecularmimicryofS.
flexneri5aO-antigen.Mostinterestingly,superimpositionoffamilyofcon-
formationsobtainedforboundp22withthisobtainedforboundDA(E)BC-
OMe shows that the type II ?-turn in the peptide seems to mimic the
nascent helicoidal shape of the oligosaccharide main chain with the aro-
matic ring of Phe4having the same orientation as the crucial branched
glucoseE(Fig.11).Thisisinagreementwithpreviousobservationsshowing
that aromatic amino acids are considered as ideal residues for mimicking
glycan side chain structures (69, 86) and that ?-turn/extended structures
may be accurate conformational mimics of helices (87, 88). Yet, except for
the number of residues involved, discriminating between the binding
modes of the three peptides remains difficult. Thus, based on binding
complementarityonly,rulesgoverningtheselectionofpotentS.flexneri5a
O-Agmimotopesremainundisclosed.Moreover,ourdataemphasizethat
therationaldesignofpeptidesmimickingtheimmunologicalpropertiesof
polysaccharidesremainsachallenge(89).Becauseinvestigatingthepeptide
bindingfeaturesleftseveralunansweredquestions,thebehaviorofthefree
peptides in solution was analyzed more closely. None of the free peptides
adopttherequiredtypeII?-turnconformationfittinginthemAbcombin-
ingsites.However,theyaresomehowpredisposedtoconformationalreor-
ganizationforbinding.Moreimportantly,p115bearingPro3andPro4both
5V. Marcel-Peyre and A. Phalipon, unpublished data.
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Page 15

involved in a turn and p100c being cyclic are both highly constrained,
whereas p22 adopting a poorly stabilized nonclassified turn is much more
flexible. Thus, our data suggest that the pre-organized conformation
adopted by the free peptide is critical for efficiency as a mimotope. It is
hypothesized that, besides the number of contacts with the mAb involved
inbinding,itmaybeespeciallyimportantthatthefreepeptideshaveastable
structurecloselyresemblingthatofthemAb-boundconformation(20,89).
Alongthislineasexemplifiedwithp22,oneofthepossibleexplanationsfor
the failure of some peptide mimics to induce a potent anti-LPS immune
response is the limited ability of small free peptides to adopt the stable
conformations necessary for functional mimicry. As a consequence, the
immune response induced is broadened resulting in the induction of low
titersofAbsexhibitingtherequiredspecificity.Toourknowledge,acrucial
issue for the development of mimotope-based vaccines remains unan-
swered, that is: what is the acceptable ability of polysaccharide-peptide
mimics to induce cross-reactive antibodies? In other words, what is an
acceptableconformationalflexibilityforthefreepeptidemimics?Ideallyifa
monovalent vaccine is the target, the range of accessible conformations
should match those of the native antigen only. In that case, induction of
cross-reactive Ab would be seen as a disadvantage, because as mentioned
above the level of specific Ab is consequently lowered. More importantly,
the risk of inducing an Ab response directed against self-antigens may be
increased. However, by allowing cross-protection against different sero-
typesofagivenbacterialspecies,cross-reactivityrelatedtocontrolledflex-
ibility may constitute an advantage if the development of multivalent vac-
cinesisenvisioned.Thisissuehastobefurtherinvestigated.
In conclusion, our work brings new data that contribute to a better
understandingofmolecularmimicryofpolysaccharideantigensbymimo-
topes. Encouraging data demonstrating the feasibility of using mimotopes
as surrogates of native antigens for the induction of protective immune
responses have been provided in the last 10 years. However, major break-
throughsarestillneededtofurtherestablishtherulesgoverningthemolec-
ularmimicryofpolysaccharideantigensbypeptides.Thoseshouldhelpthe
futuredesignofefficientmimotope-basedvaccines.
Acknowledgments—We warmly thank Franc ¸oise Baleux (Unite ´ de Chimie
Organique, Institut Pasteur, France) for the expertise, advice, and helpful dis-
cussion on peptide synthesis and purification. We also thank Audrey Thuizat
(Unite ´ de Pathoge ´nie Microbienne Mole ´culaire, Institut Pasteur, Paris) and
MoniqueReinhardt(InstitutdeBiochimie,ISREC,Epalinges,Switzerland)for
theirtechnicalsupportintheproductionandsequencingofmIgA,respectively.
The 600-MHz NMR spectrometer was funded by the Re ´gion Ile de France and
the Institut Pasteur (Paris, France).
REFERENCES
1. MacLeod, C. M., Hodges, R. G., Heidelberg, M., and Bernhard, W. G. (1945) J. Exp.
Med. 82, 445–465
2. Roy, R. (2004) Drug Discovery Today: Technologies 1, 327–336
3. Goebel, H. H., Ikeda, K., Schulz, F., Burck, U., and Kohlschutter, A. (1981) Acta
Neuropathol. 55, 247–249
4. Pozsgay, V., Chu, C., Pannell, L., Wolfe, J., Robbins, J. B., and Schneerson, R. (1999)
Proc. Natl. Acad. Sci. U. S. A. 96, 5194–5197
5. Peeters, C. C., Evenberg, D., Hoogerhout, P., Kayhty, H., Saarinen, L., van Boeckel,
C. A., van der Marel, G. A., van Boom, J. H., and Poolman, J. T. (1992) Infect. Immun.
60, 1826–1833
6. Verez-Bencomo, V., Fernandez-Santana, V., Hardy, E., Toledo, M. E., Rodriguez,
M. C., Heynngnezz, L., Rodriguez, A., Baly, A., Herrera, L., Izquierdo, M., Villar, A.,
Valdes, Y., Cosme, K., Deler, M. L., Montane, M., Garcia, E., Ramos, A., Aguilar, A.,
Medina, E., Torano, G., Sosa, I., Hernandez, I., Martinez, R., Muzachio, A., Car-
menates, A., Costa, L., Cardoso, F., Campa, C., Diaz, M., and Roy, R. (2004) Science
305, 522–525
7. Stein, H., Gatter, K. C., Heryet, A., and Mason, D. Y. (1984) Lancet 2, 71–73
8. Pirofski, L. A. (2001) Trends Microbiol. 9, 445–451
9. Johnson, M. A., Rotondo, A., and Pinto, B. M. (2002) Biochemistry 41, 2149–2157
10. Monzavi-Karbassi, B., Cunto-Amesty, G., Luo, P., and Kieber-Emmons, T. (2002)
Trends Biotechnol. 20, 207–214
11. Luo, P., Agadjanyan, M., Qiu, J., Westerink, M. A., Steplewski, Z., and Kieber-Em-
mons, T. (1998) Mol. Immunol. 35, 865–879
12. Cunto-Amesty,G.,Dam,T.K.,Luo,P.,Monzavi-Karbassi,B.,Brewer,C.F.,VanCott,
T. C., and Kieber-Emmons, T. (2001) J. Biol. Chem. 276, 30490–30498
13. Fleuridor, R., Lees, A., and Pirofski, L. (2001) J. Immunol. 166, 1087–1096
14. Lesinski, G. B., and Westerink, M. A. (2001) J. Microbiol. Methods 47, 135–149
15. Maitta, R. W., Datta, K., Lees, A., Belouski, S. S., and Pirofski, L. A. (2004) Infect.
Immun. 72, 196–208
16. Buchwald, U. K., Lees, A., Steinitz, M., and Pirofski, L. A. (2005) Infect. Immun. 73,
325–333
17. Harris,S.L.,Craig,L.,Mehroke,J.S.,Rashed,M.,Zwick,M.B.,Kenar,K.,Toone,E.J.,
Greenspan, N., Auzanneau, F. I., Marino-Albernas, J. R., Pinto, B. M., and Scott, J. K.
(1997) Proc. Natl. Acad. Sci. U. S. A. 94, 2454–2459
18. Valadon, P., Nussbaum, G., Oh, J., and Scharff, M. D. (1998) J. Immunol. 161,
1829–1836
19. Cunto-Amesty, G., Luo, P., Monzavi-Karbassi, B., Lees, A., and Kieber-Emmons, T.
(2001) Vaccine 19, 2361–2368
20. Young, A. C., Valadon, P., Casadevall, A., Scharff, M. D., and Sacchettini, J. C. (1997)
J. Mol. Biol. 274, 622–634
21. Vyas, N. K., Vyas, M. N., Chervenak, M. C., Bundle, D. R., Pinto, B. M., and Quiocho,
F. A. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 15023–15028
22. Johnson, M. A., and Pinto, B. M. (2004) Carbohydr. Res. 339, 907–928
23. Kotloff, K. L., Winickoff, J. P., Ivanoff, B., Clemens, J. D., Swerdlow, D. L., Sansonetti,
P. J., Adak, G. K., and Levine, M. M. (1999) Bull. W.H.O. 77, 651–666
24. Phalipon, A., and Sansonetti, P. J. (2003) Crit. Rev. Immunol. 23, 371–401
25. Ashkenazi, S., Passwell, J. H., Harlev, E., Miron, D., Dagan, R., Farzan, N., Ramon, R.,
Majadly, F., Bryla, D. A., Karpas, A. B., Robbins, J. B., and Schneerson, R. (1999)
J. Infect. Dis. 179, 1565–1568
26. Wright,K.,Guerreiro,C.,Laurent,I.,Baleux,F.,andMulard,L.A.(2004)Org.Biomol.
Chem. 2, 1518–1527
27. Belot,F.,Guerreiro,C.,Baleux,F.,andMulard,L.A.(2005)Chemistry11,1625–1635
28. Phalipon, A., Folgori, A., Arondel, J., Sgaramella, G., Fortugno, P., Cortese, R., San-
sonetti, P. J., and Felici, F. (1997) Eur. J. Immunol. 27, 2620–2625
29. Pincus, S. H., Lepage, S. R., Jung, R. F., Massey, J. G., and Jaseja, M. (2001) Int. Rev.
Immunol. 20, 221–227
30. Prinz, D. M., Smithson, S. L., and Westerink, M. A. (2004) J. Immunol. Methods 285,
1–14
31. Lindberg,A.A.,Cam,P.D.,Chan,N.,Phu,L.K.,Trach,D.D.,Lindberg,G.,Karlsson,
K., Karnell, A., and Ekwall, E. (1991) Rev. Infect. Dis. 13, Suppl. 4, 231–237
32. Mulard, L. A., and Ughetto-Monfrin, J. (2000) J. Carbohydr. Chem. 19, 193–220
33. Clement, M. J., Imberty, A., Phalipon, A., Perez, S., Simenel, C., Mulard, L. A., and
Delepierre, M. (2003) J. Biol. Chem. 278, 47928–47936
34. Clore, G. M., and Gronenborn, A. M. (1982) J. Magn. Reson. 48, 402–417
35. Clore, G. M., and Gronenborn, A. M. (1983) J. Magn. Reson. 53, 423–442
36. Mayer, M., and Meyer, B. (1999) Angew. Chem. Int. Ed. 38, 1784–1788
37. Johnson, M. A., and Pinto, B. M. (2002) J. Am. Chem. Soc. 124, 15368–15374
38. Johnson,M.A.,Jaseja,M.,Zou,W.,Jennings,H.J.,Copie,V.,Pinto,B.M.,andPincus,
S. H. (2003) J. Biol. Chem. 278, 24740–24752
39. Johnson, M. A., and Pinto, B. M. (2004) Bioorg. Med. Chem. 12, 295–300
40. Mulard, L. A., Cle ´ment, M.-J., Segat-Dioury, F., and Delepierre, M. (2002) Tetrahe-
dron 58, 2593–2604
41. Phalipon, A., Kaufmann, M., Michetti, P., Cavaillon, J. M., Huerre, M., Sansonetti, P.,
and Kraehenbuhl, J. P. (1995) J. Exp. Med. 182, 769–778
FIGURE11.Potentialconformationalmimetismofthepeptidep22inrelationtothe
pentasaccharide DA(E)BC-OMe. The figure shows superimposition of the p22 lowest
energystructurewiththatoftheDA(E)BC-OMe.Structuralelementsshowingthepoten-
tial conformational mimetism of the peptide in relation to the oligosaccharide are
shaded in pink.
PeptideMimicsofS.flexneri5aPolysaccharideAntigen
JANUARY 27, 2006•VOLUME 281•NUMBER 4 JOURNAL OF BIOLOGICAL CHEMISTRY 2331