Structural and physiological facets of carbohydrate-peptide mimicry were addressed by analyzing the Ab response to α-d-mannopyranoside. mAbs against α-d-mannopyranoside were generated and screened with the carbohydrate-mimicking 12 mer (DVFYPYPYASGS) peptide. Three mAbs, 2D10, 1H11, and 1H7, which were subjected to detailed analysis, exhibit diverse V gene usage, indicating their independent germline origins. Although the mAb 1H7 was specific in binding only to the immunizing Ag, the Abs 2D10 and 1H11 recognize the 12 mer peptide as well as the immunogen, α-d-mannopyranoside. The Abs that recognize mimicry appear to bind to a common epitope on the peptide and do not share the mode of peptide binding with Con A. Binding kinetics and thermodynamics of Ag recognition suggest that the Ab that does not recognize peptide-carbohydrate mimicry probably has a predesigned mannopyranoside-complementing site. In contrast, the mimicry-recognizing Abs adopt the Ag-combining site only on exposure to the sugar, exploiting the conformational flexibility in the CDRs. Although the mAb 1H7 showed unique specificity toward mannopyranoside, the mimicry-recognizing Abs 2D10 and 1H11 exhibited degenerate specificities with regard to other sugar moieties. It is proposed that the degeneracy of specificity arising from the plasticity at the Ag-combining site in a subset of the Ab clones may be responsible for exhibiting molecular mimicry in the context of Ab response.

Ensemble of independent Ab genes that can actually be generated in the primary Ab response is not unlimited even though the antigenic space is infinite (1). This dichotomy could imply molecular mimicry in the immune response. Indeed, mimicry plays a crucial role in normal and pathological functioning of biological systems that are responsible for the recognition and discrimination of self from nonself molecules. It has been widely suggested that the autoimmune disorders may be a clinical manifestation of molecular mimicry. Immune system encounters mimicry at all times, although it does not always result in autoimmunity. Elegant mechanisms have evolved for distinguishing finer molecular details that may otherwise appear similar unless there is breakdown of tolerance. The mechanisms by which the immune system addresses molecular equivalences are therefore of structural as well as functional interest. We have extensively studied the mimicry between peptides containing Tyr-Pro-Tyr motif and methyl α-d-mannopyranoside, addressing its structural basis and physiological implications by crystallographic as well as immunological methods (2, 3, 4, 5).

Con A, a lectin from Canavalis ensiformis, is known to bind the mannose-containing carbohydrates on the cell surface. In addition to the carbohydrate ligands, Con A also recognizes the peptide, DVFYPYPYASGS (12 mer). To establish the structural basis of mannopyranoside-peptide mimicry, crystal structure of the 12 mer peptide complexed with Con A was determined (4). The YPY region of the peptide exhibited significant structural similarity in terms of shape and hydropathy features with the trimannoside ligand, but it was found that the peptide did not occupy previously characterized carbohydrate binding site of Con A, providing only indirect explanations for peptide-carbohydrate mimicry. Carbohydrate-peptide mimicry was also analyzed in the context of the humoral immune response, wherein it was established, in terms of Ab cross-reactivity, and also observed during Ab maturation (2, 3). Immunization of mice with α-d-mannopyranoside gave rise to Ab response that recognized the 12 mer peptide (DVFYPYPYASGS). Correspondingly, the 12 mer peptide also led to mannopyranoside cross-reacting polyclonal Ab (pAb)4 response. Topological quasi-equivalence between the peptide and the carbohydrate moiety was implied in these observations. However, the precise molecular description of the functional mimicry between the otherwise chemically independent Ags as seen by the humoral response remained elusive. Therefore, it was apparent that the possible molecular characterization of this mimicry would require generation of mAbs.

A variety of anti-polysaccharide mAbs has been previously analyzed in the context of molecular mimicry (6, 7, 8, 9, 10). However, the structural relationship of their specificities and affinities with molecular mimicry has not been established. In this study, we have focused on the physicochemical basis of carbohydrate-peptide mimicry by analyzing Ab response against mannopyranoside at the molecular level. mAbs against mannopyranoside were generated and characterized for their binding to the carbohydrate-mimicking 12 mer (DVFYPYPYASGS) peptide. Recognition of both the carbohydrate and the peptide ligands by the mimicry-recognizing Abs with comparable affinities implied that the 12 mer peptide may be a true mimotope of the mannopyranoside Ag. It was evident that the mAbs that recognized peptide-carbohydrate mimicry showed broad specificity while exhibiting diverse idiotypic traits. Our studies suggest that the functional mimicry between the carbohydrate and the peptide ligands, as seen in the immune response, may be linked to the plasticity of the Ag-combining site for a set of Ab clones.

The conjugation of the mannopyranoside to BSA (Sigma-Aldrich, St. Louis, MO) and KLH (Sigma-Aldrich) was achieved by a two-step reaction. In the first step, p-aminophenyl-α-d-mannopyranoside (Sigma-Aldrich) was activated with an equimolar amount of glutaraldehyde (Sigma-Aldrich) in 0.1 M sodium carbonate buffer, pH 9.0, for 30 min at room temperature, and then mixed with BSA or KLH in the same buffer. The reaction mixture was incubated at 4°C overnight, after which it was extensively dialyzed against normal saline.

The 12 mer peptide was synthesized by solid-phase method on an automated peptide synthesizer (431A; Applied Biosystems, Foster City, CA) using 9-fluorenylmethyloxycarbonyl chemistry on a p-hydroxymethylphenoxymethylpolystyrene resin (Nova Biochem, San Diego, CA). Cleavage was performed using trifluoroacetic acid (Sigma-Aldrich). Crude peptide was purified on a Delta Pak C18 column (Waters, Milford, MA) using a linear gradient of acetonitrile containing 0.1% trifluoroacetic acid. The identity of the peptide was characterized by mass spectroscopy. Lysine was introduced at the C terminus of the dodecapeptide (DVFYPYPYASGS) for its conjugation to BSA using glutaraldehyde. The glutaraldehyde solution in 0.1 M phosphate buffer, pH 7.1, with 150 mM NaCl was slowly added to the cold mixture of peptide and BSA with the ratio of 100:1 up to a final concentration of 0.1% in the reaction mixture. The reaction mixture was incubated for 20 h at 4°C. Synthesized conjugate was extensively dialyzed against normal saline.

Female BALB/c mice of 6–8 wk of age were immunized with mannopyranoside-KLH. Immunizations were given with a dose of 200 μg of conjugated mannopyranoside per mouse. The mannopyranoside-KLH conjugate was allowed to adsorb on alum for ∼20 h at 4°C. The adsorbed Ag was injected i.p. in a volume of 400 μl per mouse. Animals were given a booster dose of the Ag on day 42, and the sera samples were checked for the Ab titers after 15 days of the booster. The highest responder mouse, selected for fusion, was given an i.v. injection of the mannopyranoside Ag 3 days before the mouse was sacrificed for harvesting spleen cells for the generation of hybridomas.

The sera from the immunized mice were checked for anti-mannopyranoside Ab titers. The highest responder of the group of mice was then sacrificed to harvest the B cells from the spleen. These cells were allowed to fuse with the Sp2/0 myeloma cells, maintained in log phase, in the presence of PEG1600. The cells were subjected to hypoxanthine-aminopterin-thymidine selection in DMEM (Biological Industries, Beit Haemek, Israel) in which only the hybrid cells are able to survive and grow. The supernatant of the wells with colonies of hybrid cells was screened for the presence of mannopyranoside and peptide-recognizing Ab by ELISA, in which mannopyranoside-BSA and 12 mer-BSA conjugates were used as the coating Ag, respectively. The positive clones were further subcloned using limited dilution technique to ensure monoclonality.

Eight- to 10-wk-old BALB/c mice were injected with pristane or IFA (after irradiation at 400 rad), 7–14 days before injecting hybrid cells. Cells were washed and resuspended in Dulbecco’s PBS. A total of ∼5 × 105 to 5 × 106 hybridoma cells was injected into each mouse. The ascites was tapped from the peritoneal cavity of the mice after 5–7 days. The Ab was purified from the ascites in two-step purification protocol. The first step involved precipitation of the Ab by a 40% ammonium sulfate cut. The precipitated Ab was later resolubilized in 10 mM Tris, pH 8.5, and subjected to anion exchange chromatography on DEAE column. The bound protein was eluted with a gradient of NaCl, and the purity of the Ab preparation was checked on SDS-PAGE. This preparation was used for all additional experiments. The concentration of the Ab was estimated by protein assay (Bio-Rad, Hercules, CA) using BSA as the standard.

A total of ∼1.0 × 106 to 107 hybridoma cells was used for total mRNA extraction with TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA). First strand of cDNA of both the H and the L chains of mAbs 2D10 and 1H7 and the L chain of mAb 1H11 was synthesized from total RNA (∼2 μg) using 12.5 U of reverse transcriptase (Promega, Madison, WI), 12.5 pmol 3′ primer (Mouse Ig-Primer set; Novagen, Madison, WI) and 10 mmol dNTPs in total reaction volume of 25 μl. Single-stranded cDNA was amplified using the 5′ primer (Mouse Ig-Primer set; Novagen), 25 mmol MgCl2, 10 mmol dNTPs, and 0.75 U of TaqDNA polymerase (Promega) in total reaction volume of 25 μl. The first strand cDNA of H chain of 1H11 was synthesized using 40 U of reverse transcriptase, 320 pmol 5′-GGCCAGTGGATAGAC-3′ primer, and 25 mmol dNTPs in a total reaction volume of 25 μl, starting with 4 μg of total RNA (11). Single-stranded cDNA was then amplified using 200 pmol 5′-AGGT(C/G)(A/C)A(A/G)CTGCAG(G/C)AGTC(A/T)GG-3′ as the 5′ primer and 5′-GGCCAGTGGATAGAC(T/C/A)GA-3′ as the 3′ primer, 10 mmol dNTPs, 1.5 mmol MgCl2, and 2 U of TaqDNA polymerase. PerkinElmer Thermocycler (Wellesley, MA) was used for PCR with initial denaturation at 95°C for 3 min, followed by 30 cycles each at 94°C for 1 min, 58°C for 1.5 min, 72°C for 1 min, and final incubation at 72°C for 10 min. A total of 1 μl of the PCR product was analyzed on 1% agarose gel. Subsequently, the PCR products were sequenced using their respective forward primers.

Sequentially mutating each residue of the 12 mer peptide to glycine and measuring the binding of the individual peptide analogs to the Abs would allow delineation of the Ab-specific epitope of the 12 mer peptide. The 12 mer peptide analogs were synthesized on the surface of the polyethylene pins that have been radiation grafted with acrylic acid (Pin technology; Cambridge Research Biochemicals, Cleveland, U.K.). The desired peptide synthesis is distanced from the pinhead by a molecular spacer. For synthesis, the standard protocol recommended by the manufacturer was followed using the F-moc chemistry. After completion of the synthesis, all peptide analogs were acetylated at the N terminus with 5:2:1 by volume mixture of dimethylformamide, acetic anhydride, and triethylamine, respectively. Side chain deprotection was accomplished over a 4-h period at the room temperature with a 38:1:1 by volume mixture of trifluoroacetic acid, ethanedithiol, and thioanisol, respectively. The pins with their irreversibly bound peptide analogs could be reused by removing the bound Ab by sonicating them in the recommended disruption buffer.

For evaluation of Ab binding, pins with covalently linked synthetic peptide analogs were blocked with 2% gelatin in PBS containing 0.1% Tween 20 for 2 h at 37°C. After washing, the pins were incubated with an appropriate concentration of mAb in PBS overnight at 4°C. Subsequently, pins were washed and incubated with HRP-labeled goat anti-mouse Abs for 1.5 h. The peroxidase substrate o-phenylenediamine (Sigma-Aldrich) and H2O2 were added, and the OD was measured at 490 nm after addition of 1 N H2SO4.

To assess the relative contribution of various residues of the 12 mer peptide in binding to the Ab, the change in the signal of binding to each peptide analog with reference to the native peptide was calculated. This change was expressed as percentage loss of the Ab binding with respect of the native peptide and was defined as: percentage of loss in Ab binding = ((Bnative − Banalog)/Bnative) × 100, where Bnative was the binding signal of native peptide and Banalog was the binding signal of glycine-substituted analog in the ELISA. The percentage loss in Ab binding was then plotted against each glycine-substituted residue of the 12 mer peptide.

Binding kinetics were measured by detecting surface plasmon resonance signal using IAsys Auto+ affinity biosensor (Affinity Sensor, Cambridge, U.K.). Mannopyranoside-BSA and 12 mer-BSA were covalently immobilized on carboxylate cuvettes in 10 mM sodium acetate buffer, pH 4.5 and 4.8, respectively, using amine-coupling kit supplied by the manufacturer. A total of ∼300–600 arc sec of the ligands was immobilized, in which 600 arc sec corresponds to immobilized protein concentration of 1 ng/mm2. The unreacted activated sites on the cuvettes were blocked with 1 M ethanolamine, pH 8.5. The binding studies were conducted at temperatures of 16°C, 20°C, 25°C, and 30°C. All measurements were conducted for mAb 1H7 in 10 mM HEPES, pH 7.4, containing 150 mM NaCl, 3.5 mM EDTA, and 0.05% Tween 20. For the mAbs 2D10 and 1H11, 10 mM Tris, pH 7.4, containing 150 mM NaCl, 3.5 mM EDTA, and 0.05% Tween 20, was used as the binding buffer. For the determination of association rate constants, various concentrations of Abs in binding buffer were allowed to bind to the immobilized ligands on the cuvettes. The dissociation rate constants were measured by replacing the samples with binding buffer. Regeneration was conducted using 10 mM HCl.

Association and dissociation rate constants were calculated by nonlinear fitting of the primary sensogram data using the FASTfit software package supplied with the IAsys instrument. The instrument response measured in arc seconds is proportional to the mass of bound ligate, resulting in: Rt = (Req − R0) (1 − exp (Kont)) + R0, where Rt is the response at time t, R0 is the initial response, Req the maximal response, and Kon is the pseudo-first order rate for the interaction. The response values are determined experimentally, and therefore Kon, at a particular concentration of ligate, can be derived. Multiple determinations of Kon are obtained by carrying out repeat associations at various concentrations of ligate. The value of Kon varies with ligate concentration [L] in a linear fashion: Kon = Kd + Ka [L]. A plot of multiple Kon values, derived from interaction experiment, against the ligate concentration [L] at which they were conducted, allows the determination of association constant, Ka, from the slope and dissociation rate constant, Kd, from the intercept. The dissociation rate constant, Kd, can also be measured by direct examination of dissociation data. The dissociation is observed as an exponential decay of the complex into its components, namely immobilized ligand and free ligate with time, as described by equation: Rt = R0 exp (−Kdt). The instrument response (Rt), measured in arc seconds at time t, is dependent on the initial response (R0) and the dissociation rate constant, Kd. The dissociation rate constant was determined from the dissociation data as well as from the intercept of the plot of Kon with respect to ligand concentration, to verify mutual concordance. The Kd obtained from the dissociation data were used for subsequent analysis.

The dissociation equilibrium constant, KD, can be calculated from the equation: KD = Kd/Ka.

The t1/2 of the Ab-Ag complex was calculated as t1/2 = ln2/Kd.

A binding interaction is defined by a net negative change in the Gibbs free energy of binding at equilibrium (ΔGeq), which is given by the equation ΔGeq = RTlnKD, where R is the Rydberg’s gas constant, and T is the temperature in Kelvin. ΔGeq in turn depends on individual free energy changes that accompany the association (ΔGass) and the dissociation (ΔGdiss) steps. The relationship between these three parameters is defined by the equation ΔGeq = ΔGa − ΔGd.

The energetics of either the association or the dissociation reaction results from net changes in two parameters, namely, the enthalpy (ΔH) and the entropy (ΔS). Although the enthalpy term generally describes heat changes that take place due to interactions at the binding interface, entropy changes largely represent net conformational/stereochemical/structural perturbations that occur either within the interacting entities or in the surrounding solvent molecules. Thus, the free energy changes that accompany either an association or dissociation step are defined by the following equation: ΔGa/d = ΔHa/d − TΔSa/d.

The temperature sensitivity of Ag association and dissociation rates of the two Abs was assessed on the basis of the Arrhenius plots. The slopes of the Arrhenius plots provide activation energy (Ea) for the corresponding steps. The individual thermodynamic parameters for both the association and the dissociation steps were calculated using the following equations: ΔΗa/d = Ea − RT; ln(Ka/d/T) = −ΔΗa/d/RT + TΔSa/d/R + ln(K′/h). In these equations, T represents temperature in Kelvin, R the Rydberg gas constant, K′ the Boltzmann’s constant, and h the Planck’s constant. Using these equations, the corresponding values of ΔH, TΔS, and ΔG for association and dissociation as well as the corresponding net values at equilibrium were calculated.

The binding of the various mAbs, from cell supernatant or in purified form, to the carbohydrate and peptide ligands was assayed by sandwich ELISA. The conjugate of 12 mer peptide or mannopyranoside with BSA was used as the coating Ag at a concentration of 2 μg/well on 96-well immunosorbent plates. The wells were subsequently blocked with 1% gelatin to prevent nonspecific binding, and Ab was subsequently added at appropriate dilutions. HRP-labeled goat anti-mouse IgG was used as the second Ab (Jackson ImmunoResearch, West Grove, PA), and o-phenylenediamine (Sigma-Aldrich) and H2O2 were used as peroxidase substrate. Absorbance was recorded at 490 nm after addition of 1 N H2SO4. An ELISA-based assay involving goat anti-mouse isotype alkaline phosphatase-labeled Abs (Santa Cruz Biotechnology, Santa Cruz, CA) was also used for Ab isotyping.

The protocol of the competitive ELISA included incubation of constant amounts of Ab with varying amounts of different sugars. The binding of the free Ab to the immobilized Ag was monitored by ELISA. The level of inhibition was calculated by comparison with the wells containing no inhibitor.

mAbs were generated against the α-d-mannopyranoside moiety conjugated to KLH. The immunization protocol used while analyzing the humoral response to the mannopyranoside ligand (2) was used in the present case as well. Anti-mannopyranoside Ab-secreting hybridomas were generated, as described in Materials and Methods. The positive clones were selected from among the hybridomas by screening the cell culture supernatant for anti-mannopyranoside IgG Abs. Their cross-reactivities with the 12 mer peptide were then analyzed. A large number of clones was found to recognize the mannopyranoside Ag, and many among them exhibited cross-reactivity with the 12 mer peptide as well. Twelve of the IgG-secreting clones showed different extent of binding to mannopyranoside (Fig. 1). Five of these were specific only to the carbohydrate Ag, and the remaining seven exhibited cross-reactivity with the peptide. Three mAbs, 2D10 (IgG2b), 1H11 (IgG1), and 1H7 (IgG1), were selected for detailed analysis.

FIGURE 1.

Screening of anti-mannopyranoside hybridomas with respect to carbohydrate-peptide mimicry. Twelve individual Ab-secreting clones generated against mannopyranoside were screened for binding to carbohydrate Ag (mannoside) and the 12 mer peptide mimic as detected by ELISA. The average background OD490 as seen in the controls was 0.06.

FIGURE 1.

Screening of anti-mannopyranoside hybridomas with respect to carbohydrate-peptide mimicry. Twelve individual Ab-secreting clones generated against mannopyranoside were screened for binding to carbohydrate Ag (mannoside) and the 12 mer peptide mimic as detected by ELISA. The average background OD490 as seen in the controls was 0.06.

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Binding of the three selected anti-mannopyranoside mAbs to the two ligands, mannopyranoside and the 12 mer peptide, was evaluated, at various Ab concentrations, by ELISA. The mAbs 2D10 and 1H11 showed similar binding to the carbohydrate and the 12 mer peptide ligand (Fig. 2, A and B). In contrast, mAb 1H7 showed strong binding to mannopyranoside, but did not recognize the mimicking peptide ligand (Fig. 2 C).

FIGURE 2.

Comparative reactivities of three anti-mannopyranoside mAbs for the carbohydrate Ag and its mimicking peptide. Binding of mAbs 2D10 (A), 1H11 (B), and 1H7 (C) to immobilized mannopyranoside (mannoside) and the 12 mer peptide ligand, as a function of concentration of the respective Abs analyzed by ELISA. A total of 2 μg/well p-aminophenyl-α-d-mannopyranoside conjugated to BSA and 12 mer-BSA was used as coating Ag for ELISA. The average background OD490 as seen in the controls was 0.06.

FIGURE 2.

Comparative reactivities of three anti-mannopyranoside mAbs for the carbohydrate Ag and its mimicking peptide. Binding of mAbs 2D10 (A), 1H11 (B), and 1H7 (C) to immobilized mannopyranoside (mannoside) and the 12 mer peptide ligand, as a function of concentration of the respective Abs analyzed by ELISA. A total of 2 μg/well p-aminophenyl-α-d-mannopyranoside conjugated to BSA and 12 mer-BSA was used as coating Ag for ELISA. The average background OD490 as seen in the controls was 0.06.

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The V regions of the mAbs were sequenced, and the genetic diversity among different anti-mannopyranoside Abs was analyzed. The nucleotide sequencing for both the L and the H chains of these Ab clones was conducted. Fig. 3 shows the translated amino acid sequences of the H and L chains of the various Abs.

FIGURE 3.

Analysis of V region sequences of the anti-mannopyranoside mAbs. Amino acid sequence alignment of the H (VH) and L (VL) chain V regions of the mAbs 1H7, 2D10, and 1H11. The three CDR regions of each chain are highlighted for direct correlation of sequence variations.

FIGURE 3.

Analysis of V region sequences of the anti-mannopyranoside mAbs. Amino acid sequence alignment of the H (VH) and L (VL) chain V regions of the mAbs 1H7, 2D10, and 1H11. The three CDR regions of each chain are highlighted for direct correlation of sequence variations.

Close modal

The Ig-BLAST sequence homology search (12) was conducted using the nucleotide sequences of the Abs to assign germline origins and identify relatedness, if any, among the three Abs. The germline genes thus identified for the V and J elements of the L chain and the V, D, and J elements of the H chain are shown in Table I. The L chain of mAb 2D10 had 100% identity with the bb1 V region germline sequence and 100% identity to Jκ2. The mAb 1H11 showed 99% identity to the 19-15 V region germline and 100% identity to the Jκ4. The nucleotide sequence of the L chain of the Ab 1H7 showed 97% identity to ce9 and 100% identity to Jκ1, the J region germline sequence.

Table I.

The germline origin of the anti-mannopyranoside Abs

mAbIsotypeVMDMJHCDR1CDR2CDR3mAbChainVLJLCDR1CDR2CDR3
H Chain        L Chain       
1H7 IgG1 J558.17 DSP2.2 NYWIE EILPGSGSTNYNEKFRG RGYWAYDFDY 1H7 ce9 RASQDI–––––NNYLN YTSNLHS QQGNTLPRT 
2D10 IgG2b J558.12 DFL16.1 DYIML NINPYYGSTSYNLKFKG KNYYGSSLDY 2D10 bbl RSSQSLVHSNGNTYLH KVSNRFS SQSTHVPYT 
1H11 IgG1 J558.44 DSP2.9 SYTIH YINPTSNYTNYQKFKD DGYYRAWFAY 1H11 19-15 KASQNV–––––GTNVA SASYRYS QQYNRYPFT 
mAbIsotypeVMDMJHCDR1CDR2CDR3mAbChainVLJLCDR1CDR2CDR3
H Chain        L Chain       
1H7 IgG1 J558.17 DSP2.2 NYWIE EILPGSGSTNYNEKFRG RGYWAYDFDY 1H7 ce9 RASQDI–––––NNYLN YTSNLHS QQGNTLPRT 
2D10 IgG2b J558.12 DFL16.1 DYIML NINPYYGSTSYNLKFKG KNYYGSSLDY 2D10 bbl RSSQSLVHSNGNTYLH KVSNRFS SQSTHVPYT 
1H11 IgG1 J558.44 DSP2.9 SYTIH YINPTSNYTNYQKFKD DGYYRAWFAY 1H11 19-15 KASQNV–––––GTNVA SASYRYS QQYNRYPFT 

The H chains of all three Abs originate from the various gene members of the J558 family. The H chain J558 gene family members have been previously reported in case of various other anti-polysaccharide Abs (13, 14, 15, 16). Different combinations of D and J regions have been used in the H chains of the three Ab clones. The mAb 2D10 H chain had 93% sequence identity to the VH gene J558.12, 100% sequence identity to DFL16.1, and 97% identity to JH2. Ab 1H11 showed 95% sequence identity to J558.44, 100% identity to DSP2.9, and 97% sequence identity match with the JH3. The V region of the H chain of mAb 1H7 Ab was derived from J558.17, as it showed 97% nucleotide sequence identity to it. Also, D region had 100% identity to DSP2.2, while J region was 97% identical with JH2.

The CDR-L1 showed maximum variability in terms of chain length among the three Abs. The L chain of the Ab 2D10 was longer in the CDR-L1 region in comparison with mAbs 1H11 and 1H7, although CDR-L2 and CDR-L3 were of similar lengths as the others. The mAb 2D10 had a five-residue insertion when compared with the CDR-L1 of the 1H7 and 1H11 Ab. The amino acid composition of CDR-H3 shows a preponderance of aromatic amino acids in all three Abs. The mAbs 1H7 and 2D10 had positively charged residues like arginine and lysine in the beginning of the loop and negatively charged residue like aspartic acid at the end of the CDR-H3 loop. The mAb 1H11, in contrast, showed presence of negatively charged residue (D) at the beginning of this loop and positively charged residue (R) in the central region. The differential presence of charged residues in the three Abs may give rise to unique structural features for the Ag-interacting regions, and thereby modulate the affinities and kinetics of binding of the Abs with respect to mannopyranoside and 12 mer peptide.

Having established that the three Abs are of independent germline origin, it was pertinent to explore whether they also differed in their mode of ligand recognition. Two among these Abs bind to the carbohydrate-mimicking 12 mer peptide. We therefore identified the residues of the carbohydrate-mimicking 12 mer peptide involved in direct interactions with the two mimicry-recognizing Abs. The correlation of epitope specificities among different anti-mannopyranoside mAbs was analyzed. The peptide residues recognized by Abs as compared with those involved in Con A binding were also meant to be explored. If, for a given Ab, the binding signal of a peptide analog diminished on mutating a residue to glycine, then that residue was considered to play an important role in Ab recognition and binding.

In the case of mAb 2D10 (Fig. 4,A), the two serine residues, Ser10 and Ser12, of the 12 mer peptide were found to be important for binding to the Ab in addition to the three hydrophobic residues, i.e., Phe3, Tyr6, and Tyr8, wherein Tyr6 and Tyr8 are part of the second Tyr-Pro-Tyr motif. It was evident that mAb 1H11 also recognized the same amino acid residues as mAb 2D10 (Fig. 4 B). The results of such peptide analog-binding studies indicated that both of the peptide-recognizing Abs, 2D10 and 1H11, essentially recognized same set of residues defining the epitope. The recognition pattern indicated that alternate residues in the peptide sequence were important for binding. This would suggest that the peptide may be bound to the Abs such that a face of peptide involving alternate side chains interacted with the Ab, while the other face consisting of intervening side chains may be exposed to the solvent. Thus, the five residues, Phe3, Tyr6, Tyr8, Ser10, and Ser12, form a common epitope recognized by the two Abs. However, the relative contribution of each of these residues in binding showed significant variation. Interestingly, the role of the two serine residues in binding to the Abs, apart from the Tyr-Pro-Tyr motif, had been highlighted in our earlier experiments involving pAbs (2).

FIGURE 4.

Epitope mapping of 12 mer peptide for the mimicry-recognizing mAbs using glycine-substituted analogs. The percentage loss of Ab binding to various analogs, generated by sequential mutation of each residue of the 12 mer peptide to glycine, with respect to the corresponding mutated residues of the peptide for mAbs 2D10 (A) and 1H11 (B). Higher loss in Ab binding on mutation of a particular residue would suggest that it may be critical for recognition by the Ab. The bars depict the mean percentage ± SEM.

FIGURE 4.

Epitope mapping of 12 mer peptide for the mimicry-recognizing mAbs using glycine-substituted analogs. The percentage loss of Ab binding to various analogs, generated by sequential mutation of each residue of the 12 mer peptide to glycine, with respect to the corresponding mutated residues of the peptide for mAbs 2D10 (A) and 1H11 (B). Higher loss in Ab binding on mutation of a particular residue would suggest that it may be critical for recognition by the Ab. The bars depict the mean percentage ± SEM.

Close modal

The binding kinetics of the Abs to mannopyranoside as well as the carbohydrate-mimicking 12 mer peptide was analyzed by surface plasmon resonance measurements. The detailed kinetic parameters, based on the sensograms generated at different Ab concentrations and the FASTfit plots determined at 25°C (Fig. 5), are presented in Table II. Affinities of the mAbs 1H7, 2D10, and 1H11 were within the physiological range and were similar to those observed for other anti-carbohydrate Abs binding to their respective Ags (17, 18, 19, 20). These mAbs exhibited higher affinities for the mannopyranoside ligand than that of Con A (2). The kinetic parameters (Table II) quantitatively reinforce the observation that mAb 1H7 binds only to mannopyranoside, while mAbs 2D10 and 1H11 show cross-reactivity with the 12 mer peptide with comparable affinities. Differences in the association and dissociation rate constants may be together contributing to the distinction between the sugar-specific mAb 1H7, as against the mimicry-recognizing mAbs 2D10 and 1H11. It is interesting to note that t1/2 values of complexes of both the mimicry-recognizing Abs with either mannopyranoside or the 12 mer peptide were comparable.

FIGURE 5.

Surface plasmon resonance analysis of the three mAbs for binding to the immobilized carbohydrate and peptide. The sensograms representing binding at 25°C to the immobilized carbohydrate Ag (mannoside) by mAbs 2D10 (1.6–6.4 μM) (A), 1H11 (10–60 μM) (B), and 1H7 (0.05–0.8 μM) (C) are shown in the left panel, and those to the 12 mer by mAbs 2D10 (2.5–30 μM) (D) and 1H11 (10–60 μM) (E) are shown in the middle panel. Right panel, Shows corresponding linear fit plots of Kon (s−1) as a function of Ab concentration for mAbs 2D10 (F), 1H11 (G), and 1H7 (H). Mannopyranoside-BSA and 12 mer-BSA, covalently coupled to carboxylate cuvettes, were the immobilized Ags for these experiments at concentration corresponding to 300–600 arc seconds (600 arc seconds correspond to immobilized protein concentration of 1 ng/mm2).

FIGURE 5.

Surface plasmon resonance analysis of the three mAbs for binding to the immobilized carbohydrate and peptide. The sensograms representing binding at 25°C to the immobilized carbohydrate Ag (mannoside) by mAbs 2D10 (1.6–6.4 μM) (A), 1H11 (10–60 μM) (B), and 1H7 (0.05–0.8 μM) (C) are shown in the left panel, and those to the 12 mer by mAbs 2D10 (2.5–30 μM) (D) and 1H11 (10–60 μM) (E) are shown in the middle panel. Right panel, Shows corresponding linear fit plots of Kon (s−1) as a function of Ab concentration for mAbs 2D10 (F), 1H11 (G), and 1H7 (H). Mannopyranoside-BSA and 12 mer-BSA, covalently coupled to carboxylate cuvettes, were the immobilized Ags for these experiments at concentration corresponding to 300–600 arc seconds (600 arc seconds correspond to immobilized protein concentration of 1 ng/mm2).

Close modal
Table II.

Affinity parameters for the interaction of the three mAbs with the carbohydrate and the peptide ligand at 25°C

LigandMannopyranosideDVFYPYPYASGS
mAbsKa × 10−5 (M−1s−1)Kd × 102 (s−1)KD (μM)t1/2 (s)Ka × 10−5 (M−1s−1)Kd × 102 (s−1)KD (μM)t1/2 (s)
1H7 1.38 ± 1.01 2.41 ± 0.17 0.17 28.8     
2D10 0.84 ± 0.13 29.72 ± 1.16 3.54 2.3 0.30 ± 0.04 29.72 ± 1.16 8.08 2.9 
1H11 0.11 ± 0.01 38.99 ± 1.13 35.45 1.8 0.17 ± 0.01 36.69 ± 1.38 21.58 1.9 
LigandMannopyranosideDVFYPYPYASGS
mAbsKa × 10−5 (M−1s−1)Kd × 102 (s−1)KD (μM)t1/2 (s)Ka × 10−5 (M−1s−1)Kd × 102 (s−1)KD (μM)t1/2 (s)
1H7 1.38 ± 1.01 2.41 ± 0.17 0.17 28.8     
2D10 0.84 ± 0.13 29.72 ± 1.16 3.54 2.3 0.30 ± 0.04 29.72 ± 1.16 8.08 2.9 
1H11 0.11 ± 0.01 38.99 ± 1.13 35.45 1.8 0.17 ± 0.01 36.69 ± 1.38 21.58 1.9 

The effects of temperature on the binding kinetics of the two mAbs, 1H7 and 2D10, with the Ag anti-d-mannopyranoside were also studied. In addition to the data at 25°C, the association and the dissociation rate constants of the mAbs binding to mannopyranoside were determined at 16, 20, and 30°C (Table III). Fig. 6, A and B, represents the linear fit plots of Kon vs Ab concentration at various temperatures for mAbs 2D10 and 1H7, respectively. The variation in Ka and Kd as a function of temperature is plotted in Fig. 6, C and D, for both of these Abs. An increase in temperature from 16°C to 30°C resulted in no significant change in the rate of association or dissociation for the mAb 1H7. In contrast, there was a marked decrease in Ka value with increase in temperature in the case of mAb 2D10, and a concomitant increase in Kd (Fig. 6, C and D). Fig. 6, E and F, represents the Arrhenius plots for both of the mAbs. Thus, the two kinetic parameters are highly sensitive to the temperature changes in case of Ab 2D10, but not in case of mAb 1H7. Correspondingly, there was no significant change in the affinity of the Ab 1H7 toward mannopyranoside as against a marked decrease in the affinity of mAb 2D10 toward the Ag with increasing temperatures.

Table III.

Affinity parameters of the mAbs 1H7 and 2D10 at four different temperatures for the carbohydrate ligand

mAb1H72D10
Temp.Ka × 10−5 (M−1s−1)Kd × 102 (s−1)KD (μM)t1/2 (s)Ka × 10−5 (M−1s−1)Kd × 102 (s−1)KD (μM)t1/2 (s)
16°C 1.68 ± 0.15 2.08 ± 0.21 0.12 34.7 1.30 ± 0.21 6.22 ± 0.39 0.48 11.1 
20°C 1.29 ± 0.17 2.09 ± 0.10 0.16 33.1 1.21 ± 0.23 21.62 ± 1.31 1.79 3.2 
25°C 1.38 ± 1.01 2.41 ± 0.17 0.17 28.8 0.84 ± 0.13 29.72 ± 1.16 3.54 2.3 
30°C 1.34 ± 0.11 6.30 ± 0.16 0.47 11.0 0.29 ± 0.03 36.75 ± 3.77 12.67 1.9 
mAb1H72D10
Temp.Ka × 10−5 (M−1s−1)Kd × 102 (s−1)KD (μM)t1/2 (s)Ka × 10−5 (M−1s−1)Kd × 102 (s−1)KD (μM)t1/2 (s)
16°C 1.68 ± 0.15 2.08 ± 0.21 0.12 34.7 1.30 ± 0.21 6.22 ± 0.39 0.48 11.1 
20°C 1.29 ± 0.17 2.09 ± 0.10 0.16 33.1 1.21 ± 0.23 21.62 ± 1.31 1.79 3.2 
25°C 1.38 ± 1.01 2.41 ± 0.17 0.17 28.8 0.84 ± 0.13 29.72 ± 1.16 3.54 2.3 
30°C 1.34 ± 0.11 6.30 ± 0.16 0.47 11.0 0.29 ± 0.03 36.75 ± 3.77 12.67 1.9 
FIGURE 6.

Kinetic analysis of the binding of mAbs 2D10 and 1H7 to the immobilized carbohydrate as a function of temperature. The linear fit plots of Kon (s−1) as a function of various concentrations of mAbs 2D10 (A) and 1H7 (B) at 16°C, 20°C, 25°C, and 30°C. The correlation coefficient for each fit was >0.96. Plots of variation of Ka (C) and Kd (D) with temperature for mAbs 2D10 and 1H7. Arrhenius plots (plot of natural log of association (E) and dissociation (F) rate constants as a function of the reciprocal of temperature) for mAbs 2D10 and 1H7. Mannopyranoside-BSA, covalently coupled to carboxylate cuvettes, was the immobilized Ag.

FIGURE 6.

Kinetic analysis of the binding of mAbs 2D10 and 1H7 to the immobilized carbohydrate as a function of temperature. The linear fit plots of Kon (s−1) as a function of various concentrations of mAbs 2D10 (A) and 1H7 (B) at 16°C, 20°C, 25°C, and 30°C. The correlation coefficient for each fit was >0.96. Plots of variation of Ka (C) and Kd (D) with temperature for mAbs 2D10 and 1H7. Arrhenius plots (plot of natural log of association (E) and dissociation (F) rate constants as a function of the reciprocal of temperature) for mAbs 2D10 and 1H7. Mannopyranoside-BSA, covalently coupled to carboxylate cuvettes, was the immobilized Ag.

Close modal

Changes in the Gibbs free energy (ΔGeq) of binding, as a function of temperature, are shown in Fig. 7,A. Whereas the change in ΔGeq in case of mAb 1H7 was ∼2 kJ/mol, the corresponding change for mAb 2D10 was ∼7 kJ/mol, consistent with the changes in the affinities of the two mAbs with respect to temperature. Causes of the differential effects of temperature on Ag binding by the two Abs were further analyzed by calculating changes in enthalpy (ΔH), entropy (TΔS), and Gibbs free energy (ΔG) at 30°C (Fig. 7, B–D). With regard to the enthalpy contributions for mAb 2D10, highly favorable changes during association were substantiated by large unfavorable changes during dissociation. Although the trends were similar, significantly lower changes in enthalpy were observed for Ab 1H7, during association as well as dissociation steps. Effectively, this resulted in larger favorable ΔHeq for mAb 2D10 than for mAb 1H7.

FIGURE 7.

Thermodynamic analysis of binding to immobilized carbohydrate for mAbs 2D10 and 1H7. The comparison of the Gibbs free energy of binding to mannopyranoside by the two Abs 1H7 and 2D10 at different temperatures (A), change in enthalpy (B), and entropy (TΔS) (C) values for the association and dissociation phase of binding for mAbs 2D10 and 1H7 at 30°C. The values were calculated from the Arrhenius plots, as described in Materials and Methods. Schematic representation of the reaction profile in terms of Gibbs free energy changes for mAbs 2D10 and 1H7 (D) at 30°C.

FIGURE 7.

Thermodynamic analysis of binding to immobilized carbohydrate for mAbs 2D10 and 1H7. The comparison of the Gibbs free energy of binding to mannopyranoside by the two Abs 1H7 and 2D10 at different temperatures (A), change in enthalpy (B), and entropy (TΔS) (C) values for the association and dissociation phase of binding for mAbs 2D10 and 1H7 at 30°C. The values were calculated from the Arrhenius plots, as described in Materials and Methods. Schematic representation of the reaction profile in terms of Gibbs free energy changes for mAbs 2D10 and 1H7 (D) at 30°C.

Close modal

The favorable ΔHeq is severely attenuated by almost equally large unfavorable entropic changes for mAb 2D10, but not so in the case of mAb 1H7. Although the changes in the entropy component during association (TΔSass) were highly unfavorable, those during dissociation (TΔSdiss) were marginally favorable for mAb 2D10. In contrast, the entropy changes during both association as well as dissociation steps were small and unfavorable for Ab 1H7. Although binding to the Ag is enthalpically driven for both the Abs, the high unfavorable entropic contribution resulted in less favorable ΔGeq for mAb 2D10. In comparison, ΔGeq was more favorable in the case of the Ab 1H7, even though changes in enthalpy and entropy components were not as dramatic. This has been qualitatively illustrated by plotting reaction profiles of the two Abs at 30°C (Fig. 7 D). The substantially more negative ΔGeq value for Ab 1H7 is contributed by both lower ΔGass and higher ΔGdiss. This effect is also reflected in the >25-fold higher affinity of mAb 1H7 as compared with mAb 2D10 for the mannopyranoside ligand at 30°C.

The anti-carbohydrate as well as the anti-peptide pAb response was shown to be specifically directed against the mannopyranoside. It was evident that both antisera cross-reacted poorly with other related sugars (2). Although mannopyranoside is the specific native Ag, glucopyranoside is a different, but related carbohydrate. Lactose, a disaccharide, was considered as a nonspecific carbohydrate moiety. We investigated the extent of specificity associated with the different mAbs by comparing their binding with the above mentioned carbohydrate ligands. Competitive ELISA-based assays were conducted, in which the decrease in binding of the Ab to the immobilized ligand in the presence of various concentrations of competing sugars was calculated as the inhibition due to these sugars.

All of the carbohydrate moieties, mannopyranoside, glucopyranoside, and lactose, showed similar competition profiles for the mimicry-recognizing Abs, 2D10 and 1H11 (Fig. 8, A and B). In case of mAb 1H7, although the binding of Ab to immobilized mannopyranoside was inhibited by the soluble mannopyranoside, glucopyranoside did not compete to a comparable extent and lactose showed no inhibition (Fig. 8 C). Thus, it is evident that mannopyranoside is a specific ligand of mAb 1H7.

FIGURE 8.

Competitive inhibition profiles of the Abs for binding to immobilized carbohydrate and peptide by different sugars in solution. Plots representing competition for binding to immobilized mannopyranoside Ag by mannopyranoside (mannoside), glucopyranoside (glucoside), and lactose in solution for mAbs 2D10 (A), 1H11 (B), and 1H7 (C). Competitive inhibition profiles of 2D10 (D) and 1H11 (E) binding to the immobilized 12 mer ligand by the same sugars. The plot shows percentage of decrease in binding to the immobilized ligand by the Abs, as detected by ELISA, in the presence of various concentrations of competing sugars.

FIGURE 8.

Competitive inhibition profiles of the Abs for binding to immobilized carbohydrate and peptide by different sugars in solution. Plots representing competition for binding to immobilized mannopyranoside Ag by mannopyranoside (mannoside), glucopyranoside (glucoside), and lactose in solution for mAbs 2D10 (A), 1H11 (B), and 1H7 (C). Competitive inhibition profiles of 2D10 (D) and 1H11 (E) binding to the immobilized 12 mer ligand by the same sugars. The plot shows percentage of decrease in binding to the immobilized ligand by the Abs, as detected by ELISA, in the presence of various concentrations of competing sugars.

Close modal

In a similar experiment, various sugars were made to compete with the immobilized 12 mer peptide for the Ab binding. The inhibition was brought about by different sugars in the binding of two mimicry-recognizing Abs, 2D10 and 1H11, to the immobilized 12 mer peptide shown in Fig. 8, D and E. In both cases, the inhibition profiles generated by various sugars were similar to that observed in case of immobilized mannopyranoside.

Molecular mimicry associated with mannopyranoside and 12 mer peptide was earlier established by analyzing the corresponding pAb responses and structures of peptide-Con A complexes (2, 3, 4, 5, 21, 22). Structural analyses of the complexes of peptide ligands with Con A provided topological correlation between the 12 mer peptide and the mannopyranoside ligand (5, 21). The analysis of carbohydrate-peptide mimicry using anti-mannopyranoside pAbs had shown a high degree of specificity for the immunizing Ag (2). Mannopyranoside showed much higher inhibition of pAb binding to immobilized mannopyranoside as compared with other sugars, including glucopyranoside and lactose. In other words, the anti-mannopyranoside pAb response revealed fine distinction between different sugars competing against mannopyranoside, but lacked similar ability to discriminate when competed against the 12 mer peptide. Thus, the humoral immune response was thought to be a rather heterogeneous pool of independent Abs with different specificities.

To establish molecular basis of the peptide-carbohydrate mimicry, we set out to characterize the mimicry at the level of individual Ab clones. Rationale for this approach was essentially to pick Ab clones that bind to the immunizing Ag, α-d-mannopyranoside, and subsequently screen them for the ability to recognize the mimicking peptide. An alternate approach could involve screening and characterization of hybridomas generated against 12 mer for binding to the mannopyranoside ligand. Thus, the mAbs that were specific to the sugar as well as those that recognized peptide-carbohydrate mimicry were generated from the anti-mannopyranoside response. Nearly half among the 12 clones characterized exhibited binding to the carbohydrate-mimicking peptide. Three anti-mannopyranoside mAbs, two mimicry recognizing (2D10 and 1H11) and one noncross-reacting (1H7), were extensively characterized. The V region sequences of the anti-mannopyranoside mAbs exhibited differences in the CDRs. The predicted germline origins of the H and L chains of the three clones suggested that they have arisen from independent progenitor B cells and have not evolved due to somatic mutations in the Ig gene of a single B cell. It can thus be inferred that the three mAbs against mannopyranoside have independent germline origins.

The epitope-mapping data qualitatively implied that the Abs recognizing peptide-carbohydrate mimicry bind to a common epitope on the 12 mer peptide. The two mimicry-recognizing mAbs, 2D10 and 1H11, recognize alternate amino acids spread over the entire sequence. In contrast, Con A primarily recognizes the Tyr-Pro-Tyr motif and its flanking residues (4, 5). It was shown that the anti-mannopyranoside pAbs exhibited Con A-like fine specificity in carbohydrate recognition (2), and that the sugar and the sugar-mimicking peptide bind to Con A with comparable affinities (23). These observations together could imply possibility of a common ligand-recognition mechanism, perhaps through similar binding interfaces, for Con A and anti-mannopyranoside Abs. However, that was certainly not the case. The mode of 12 mer peptide binding to the anti-mannopyranoside mAbs appears to be very different from the binding of carbohydrate-mimicking peptides to Con A (4, 21). In other words, the mimicry-recognizing anti-mannopyranoside mAbs do not represent an internal image of Con A. Thus, the genetically independent mimicry-recognizing mAbs share a common epitope on the carbohydrate-mimicking peptide among themselves that is different from that of Con A.

The independent mimicry-recognizing mAbs sharing common epitope on the carbohydrate-mimicking peptide could imply structural incongruity. In ligand-receptor recognition, it is expected that if two ligands are structurally similar, then the ligand binding sites of their receptors also exhibit structural similarity (24, 25). The restricted V gene usage in the anti-carbohydrate response has been used to imply similar recognition of a single carbohydrate epitope by various Abs (26). In the present set of anti-mannopyranoside mAbs, the mimicry-recognizing as well as mannopyranoside-specific mAbs do not show any kind of V gene restriction among themselves, implying variable paratope structures. Despite the obvious differences in the paratopes, the two mimicry-recognizing Abs bind to the peptide through a common set of residues, as is evident from the epitope-mapping data. In other words, the anti-mannopyranoside Abs recognize a common epitope despite the variations in the paratopes of these Abs. Similarity of epitope, but differences in the paratope implicated plasticity to play a role in the Ag-Ab recognition, such that the Abs could structurally adapt for binding to somewhat similar ligands (27, 28, 29).

The mAbs recognizing molecular mimicry exhibited affinities for 12 mer peptide and the mannopyranoside ligand well within the range of physiological relevance (17, 18, 19, 20). Kinetic analysis of Ag binding to the three anti-mannopyranoside mAbs exhibited differences in terms of their Ka and Kd, suggesting possible implications at the structural level. Relatively fast association and substantially slow dissociation rates of mAb 1H7 in comparison with mAbs 2D10 and 1H11 could imply that the Ab 1H7 binding site may be predesigned for the carbohydrate Ag, while that of mAbs 2D10 and 1H11 might be required to undergo conformational changes while binding to the Ag.

The conformational flexibility of mAb 2D10 was also manifested as increase in dissociation rates on increasing the temperature, thereby weakening the binding to the mannopyranoside ligand. Larger changes as a function of temperature in both Ka and Kd suggest a flexible/loose fit of the ligand in the Ag-combining site in the case of mAb 2D10. Thus, the KD of mAb 1H7 undergoes small change from 16 to 30°C in comparison with that of Ab 2D10, for which it increases by >25-fold. Thermodynamic analyses of Ag binding to both mAbs are also consistent with the structural interpretations based on the kinetic data. Changes as a function of temperature in the equilibrium-free energy were substantial for mAb 2D10 in comparison with mAb 1H7. Although binding to the mannopyranoside ligand was enthalpically driven for both mAbs, the entropy contributions were significantly different. In the case of mAb 2D10, TΔSass during association was highly unfavorable, and that during dissociation (TΔSdiss) was marginally favorable. In contrast, the entropy changes during both association as well as dissociation steps were unfavorable and significantly smaller in the case of mAb 1H7. The proposition that while the Ab 1H7 may have a predefined fit for binding to the immunogen, the mimicry-recognizing mAb 2D10 possesses conformational flexibility in the CDRs, is thus reinforced. Therefore, it can be inferred that mAbs 2D10 and 1H11 adopt toward accommodating the sugar through the plasticity of interactions brought about by the flexibility of the combining site. This conformational adaptation of the flexible combining sites could also be the reason for the Abs to recognize the mannopyranoside-mimicking 12 mer peptide as well.

Among the Abs characterized, the mAb 1H7, which did not recognize mimicry, exhibited fine specificity akin to that observed in the case of pAb response. This Ab was also shown to be thermodynamically less flexible. In contrast, the two mimicry-recognizing Abs, 2D10 and 1H11, exhibited degeneracy of specificity such that the Abs failed to show discrimination among mannopyranoside, glucopyranoside, and lactose. This is contrary to the expectations, considering that the pAb response against mannopyranoside exhibited explicit specificity in favor of mannopyranoside (2). However, this is consistent with the inference, based on temperature-dependent affinity measurements, that the Ab may exhibit conformational flexibility in the Ag-combining site that could account for its ability to recognize multiple ligands.

The extent of plasticity associated with an Ag-combining site of an Ab could have implications to the corresponding binding specificity. Plasticity could provide wider binding repertoire, a necessary requirement for independent ligands to bind to a common site. The mannopyranoside-mimicking 12 mer peptide and mannopyranoside, although chemically dissimilar, are not topologically unrelated, as seen from the comparative crystallographic studies (5). It was shown that molecular mimicry between the chemically unrelated ligands does not require complete structural equivalence; instead, the quasi-equivalence involving critical structural properties may adequately provide physiologically effective molecular mimicry (4, 5, 21, 30). This was particularly evident in our earlier design of T cell epitope mimics (31). In our present study, the anti-carbohydrate Ab that does not recognize peptide seems to be least flexible with a rigid and preformed carbohydrate binding site, thereby restricting the ligand recognition to the mannopyranoside sugar. In contrast, the flexibility associated with the CDR conformations may provide plasticity to recognize other carbohydrate moieties as well as the mannopyranoside-like peptides.

In summary, we have analyzed the molecular mimicry as seen by the humoral Ab response and characterized a finite number of individual Ab clones from polyclonal population toward establishing the basis of molecular mimicry as seen by the immune system. It was evident that only those mAbs that do not recognize mimicry exhibit fine specificity and probably have predesigned sugar-complementing site, while the Abs that recognize mimicry adopt a sugar-specific site only on exposure to the sugar. The humoral Ab response as a whole relates to an internal image of Con A, but not the individual mAbs. However, a variety of different structural properties may be exploited for facilitating mimicry depending on the context. In other words, degeneracy of specificity and plasticity of interactions are indeed associated with the humoral Ab response and could exemplify molecular mimicry, as observed in our studies.

We gratefully acknowledge assistance from Sushma Nagpal and K. K. Sarin and discussions with Drs. Ayub Qadri and K. V. S. Rao.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

The work was supported by Department of Biotechnology, Government of India.

4

Abbreviations used in this paper: pAb, polyclonal Ab; KLH, keyhole limpet hemocyanin.

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