Topological Analysis of the Functional Mimicry between a Peptide and a Carbohydrate Moiety*

(Received for publication, August 1, 1996, and in revised form, November 5, 1996)

Kanwal J. Kaur , Sumit Khurana and Dinakar M. Salunke Dagger

From the Structural Biology Unit, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

The shared surface topology of two chemically dissimilar but functionally equivalent molecular structures has been analyzed. A carbohydrate moiety (alpha -D-mannopyranoside) and a peptide molecule (DVFYPYPYASGS) bind to concanavalin A at a common binding site. The cross-reactivity of the polyclonal antibodies (pAbs) was used for understanding the topological relationship between these two independent ligands. The anti-alpha -D-mannopyranoside pAbs recognized various peptide ligands of concanavalin A, and the anti-DVFYPYPYASGS pAbs recognized the carbohydrate ligands, providing direct evidence of molecular mimicry. On the basis of differential binding of various rationally designed peptide analogs to the anti-alpha -D-mannopyranoside pAbs, it was possible to identify different peptide residues critical for the mimicry. The comparison of circular dichroism profiles of the designed analogs suggests that the carbohydrate mimicking conformation of the peptide ligand incorporates a polyproline type II structural fold. The concanavalin A binding activity of these analogs was found to have a direct correlation with the topological relationship between peptide and carbohydrate ligands.


INTRODUCTION

The functional mimicry involving unrelated molecules is often encountered. Many times it occurs by design and is used as an effective control during various regulatory mechanisms. However, sometimes the accidental structural resemblances lead to aberrations, which are expressed in terms of clinical manifestations. For example, it has been suggested that the mimicry between microbial or viral peptides and the self-peptides presented inappropriately on a target tissue could initiate an autoimmune attack (1). On the other hand, many enzymatically regulated events are controlled by intervention of proteinaceous inhibitors, which mimic substrate binding. The complementarity between serine proteases and the corresponding substrates is matched by aprotinin, the chymotrypsin inhibitor, and many other such inhibitors (2, 3). In addition, molecular mimicry has implications for rational drug design applications. Considerable efforts have been invested in the development of peptidomimetic drugs using nonpeptidyl surrogates (4-7).

Systematic approaches involving computational as well as experimental tools have been used to analyze and exploit topological similarity between dissimilar molecules (8-12). It is apparent that the structural rules governing molecular mimicry are required to be defined for its successful exploitation. Concanavalin A (ConA),1 a lectin known to be specific for binding to certain mannose-containing carbohydrates, provides an appropriate model system for this purpose. Peptidyl ligands have been characterized that bind to ConA with affinities comparable with those of the carbohydrate ligand methyl alpha -D-mannopyranoside (13, 14). We have shown that the peptide and the carbohydrate ligands of ConA are true topological mimics. Cross-reactivity of polyclonal antibodies (pAbs) against an assortment of designed peptide analogs is used here to delineate the specific residues that may be contributing to the mimicry of carbohydrate structure by the peptide ligand.


MATERIALS AND METHODS

Conjugation of Mannose with BSA or DT

Conjugation of mannose with bovine serum albumin (BSA; Sigma) or diphtheria toxoid (DT; Serum Institute) was achieved by a two step reaction. In the first step, p-aminophenyl-alpha -D-mannopyranoside (Sigma) was activated by an equimolar amount of glutaraldehyde (Sigma) in 0.1 M sodium carbonate buffer, pH 9.0, for 30 min at 20 °C and then mixed with BSA or DT in the same buffer. Synthesized conjugate was extensively dialyzed against normal saline at 4 °C. The carbohydrate content of the conjugate was determined by the phenol-sulfuric acid assay (15), and its protein concentration was measured by the Bradford assay (16) using BSA as a standard. Mannopyranoside-BSA or -DT conjugate was prepared using a molar ratio of 50 mol of sugar:1 mol of BSA or DT.

Peptide Synthesis and Conjugation to BSA or DT

Assembly of the protected peptide chains was carried out on an automated peptide synthesizer, (431A, Applied Biosystems Inc.) using solid phase 9-fluorenylmethyloxycarbonyl chemistry on a p-hydroxymethyl phenoxymethyl polystyrene resin (Nova Biochem). All amino acids (Nova) were protected at the alpha -amino position with the 9-fluorenylmethyloxycarbonyl group. Cleavage was performed by using trifluoroacetic acid (Sigma). The peptides were purified on a Delta Pak C18-100Å column (19 mm × 30 cm, 15 µm, spherical; Waters) using a linear gradient of trifluoroacetic acid and acetonitrile. The absorption was monitored at 214 nm. Lysine was introduced at the carboxyl terminus of the dodecapeptide (DVFYPYPYASGS) for its conjugation to BSA or DT using glutaraldehyde. The glutaraldehyde solution in 0.1 M phosphate buffer, pH 7.1, containing 150 mM NaCl was slowly added to the cold mixture of peptide and BSA or DT with the ratio of 100:1 in the same buffer. The final concentration of glutaraldehyde was 0.1% in the reaction mixture. The reaction mixture was incubated for 20 h at 4 °C. Synthesized conjugate was exhaustively dialyzed against normal saline at 4 °C.

Immunization of Mice

BALB/c mice were immunized intraperitoneally with alpha -D-mannopyranoside-BSA or peptide (12 mer)-DT in alum-adsorbed 100 µg of protein. Mice received three injections at intervals of 2 weeks and were bled from the retro-orbital venous plexus 15 days after the last injection. The normal mice sera were obtained from mice before the first injection. Collected sera were stored in aliquots at -20 °C.

ELISA

To carry out ELISA, the corresponding conjugate of peptide (1 µg/well) or sugar (5 µg/well) with DT or BSA was absorbed on Nunc (Roskilde, Denmark) immunoplates. After coating, wells were blocked with 1% BSA or gelatin to prevent nonspecific binding, before adding serial dilutions of mouse pAbs. The polyclonal antisera were used as such for the ELISA experiments. Horseradish peroxidase-labeled goat anti-mouse Abs (NII reagent bank) were used as second antibodies. The peroxidase substrate was o-phenylenediamine (Sigma). The color was developed using H2O2, and absorbance was recorded at 490 nm.

The general protocol of the competitive ELISA included incubation of constant amounts of pAbs with equal volumes of increasing concentrations of peptides. Binding of free pAbs to absorbed antigen was measured by ELISA. The levels of inhibition were calculated by comparison of the absorbance at 490 nm of the wells reacted with pAbs and soluble inhibitor and that of the control wells, which did not contain the inhibitor. The concentrations of pAbs used in all the inhibition assays were such that the absorbance values at 490 nm were in the range of 0.8 and 1.0 for the wells without inhibitor.

Concanavalin A Binding Assay

Plates with preabsorbed antigen were incubated with biotinylated ConA (Sigma) in 50 mM phosphate, pH 7.1, containing 150 mM NaCl, 10 nM MnCl2, 100 nM CaCl2, and 0.1% Tween 20 for 1.5 h at 37 °C. The binding of ConA was detected by streptavidin-peroxidase (Vector Laboratories). As controls, wells coated with the same antigen and incubated with streptavidin-peroxidase without lectin were used. The procedure of competitive lectin binding assay was identical to the competitive ELISA.

Circular Dichroism

The circular dichroism (CD) experiments were carried out on a JASCO 710 spectropolarimeter with a 2.0-nm bandwidth, 1-nm resolution, and 1-s response time. A 10-mm-path length cell was used. Typically, 10 scans at a scan speed of 50 nm/min were added and averaged. Peptide concentrations were 50 µM in H2O. Results are expressed as molar ellipticity in deg cm2/dmol.


RESULTS

Relationship between Peptide and Carbohydrate Ligands of ConA

ConA binds to the dodecapeptide DVFYPYPYASGS (12-mer), as shown in Fig. 1A. A glycan ligand of ConA (17), methyl alpha -D-mannopyranoside, competitively inhibited this binding (Fig. 1B). This confirmed that methyl alpha -D-mannopyranoside sterically interferes in the ConA peptide binding in a dose-dependent manner as suggested earlier (13, 14). Thus, the carbohydrate binding site and the peptide binding site are overlapping.


Fig. 1. Peptide ligand binds to ConA at its carbohydrate binding site. A, reaction of biotinylated ConA with 12-mer DT in the lectin binding assay. B, inhibition of ConA binding to solid phase 12-mer DT by soluble methyl alpha -D-mannopyranoside in the competitive lectin binding assay. Biotinylated ConA in a concentration of 4 µg/ml was incubated with an increasing amount of methyl alpha -D-mannopyranoside, and binding of free biotinylated lectin to absorbed 12-mer DT was measured using peroxidase-labeled streptavidin.
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The murine pAbs were raised against alpha -D-mannopyranoside using the glycan moiety conjugated to BSA. The reactivity of these pAbs to alpha -D-mannopyranoside-DT conjugate in the ELISA confirmed that the antisera had alpha -D-mannopyranoside-specific antibodies (Fig. 2A). The control wells coated with DT showed a negligible signal, suggesting that there was no nonspecific binding. Similarly, the pAbs raised against 12-mer DT showed binding to 12-mer BSA but not to BSA, confirming that the antisera had 12-mer peptide-specific antibodies (Fig. 2B). The control sera obtained from unimmunized mice did not show any binding to either the carbohydrate ligand alpha -D-mannopyranoside (Fig. 2A) or the ligand 12-mer peptide (Fig. 2B). The cross-reactivities of the sugar and peptide pAbs were analyzed (Fig. 2C). ELISA carried out using various dilutions of the anti-12-mer pAbs showed that the antibodies recognize the glycan moiety, alpha -D-mannopyranoside. Conversely, the ELISA carried out using similar dilutions of the anti-alpha -D-mannopyranoside antibodies showed activity against the 12-mer peptide.


Fig. 2. The cross-reactivity of specific pAbs of the carbohydrate and the peptide ligands of ConA suggests structural mimicry. A, direct binding ELISA analyzing the reactivity of murine anti-alpha -D-mannopyranoside-BSA pAbs with alpha -D-mannopyranoside-DT and DT at increasing dilutions of the pAbs. Also shown are the control data indicating negligible binding of the normal mice sera to alpha -D-mannopyranoside-DT. B, direct binding assay analyzing reactivity of murine anti-12-mer DT pAbs to 12-mer BSA and BSA. The control data showing negligible binding of normal mice sera to 12-mer BSA are also shown. C, reaction of mouse anti-12-mer DT pAbs with alpha -D-mannopyranoside-BSA and the anti-alpha -D-mannopyranoside-BSA pAbs with 12-mer DT in the ELISA at increasing dilutions of the pAbs.
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Comparison between Different Carbohydrate Moieties

The specificity of pAbs against the sugar moiety, alpha -D-mannopyranoside, was further confirmed by competitive ELISA against various sugars, as shown in Fig. 3A. At a 500 mM concentration, methyl alpha -D-glucopyranoside showed less than half the activity of the specific antigen methyl alpha -D-mannopyranoside. No cross-reactivity of these pAbs was observed against alpha -lactose. Thus, the antibodies raised against alpha -D-mannopyranoside showed structurally correlated differential binding to different sugar moieties.


Fig. 3. The anti-alpha -D-mannopyranoside-BSA and the anti-12-mer DT pAbs preferentially bind to alpha -D-mannopyranoside. A, competitive inhibition of anti-alpha -D-mannopyranoside pAbs binding to solid phase alpha -D-mannopyranoside-DT by soluble methyl alpha -D-mannopyranoside, methyl alpha -D-glucopyranoside, and alpha -lactose. B, Competitive inhibition of anti-12-mer DT pAbs binding to solid phase alpha -D-mannopyranoside-BSA by soluble methyl alpha -D-mannopyranoside, methyl alpha -D-glucopyranoside, and alpha -lactose. Constant amounts of pAbs were incubated with increasing amounts of soluble methyl alpha -D-mannopyranoside, methyl alpha -D-glucopyranoside, or alpha -lactose, and binding of free pAbs to absorbed alpha -D-mannopyranoside-BSA or -DT was measured by ELISA.
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Similarly, the polyclonal antisera against the peptide molecule also distinguished finer details of the topological relationship between different carbohydrate molecules. It is known that ConA recognizes methyl alpha -D-glucopyranoside with an affinity lower than that for methyl alpha -D-mannopyranoside (18). The comparative binding of antipeptide pAbs to methyl alpha -D-glucopyranoside, methyl alpha -D-mannopyranoside, and alpha -lactose was analyzed. It was found that anti-12-mer pAbs exhibited binding to methyl alpha -D-mannopyranoside better than to methyl alpha -D-glucopyranoside and did not recognize alpha -lactose (Fig. 3B). About a four times lower concentration of the methyl alpha -D-mannopyranoside was required than that of methyl alpha -D-glucopyranoside for achieving 30% inhibition. The difference between the two sugars in their antipeptide pAb binding profile is comparable with the difference in their ConA binding.

Comparison between Different Peptide Analogs

Different peptide ligands of ConA were analyzed using polyclonal antibodies against the carbohydrate ligand alpha -D-mannopyranoside. Besides the 12-mer peptide, two other peptides, MYWYPY (6- mer) and RVWYPYGSYLTASGS (15 mer), have also been identified to bind to ConA with affinities comparable with that of the carbohydrate ligand (13, 14). The binding affinity of antisugar pAbs to these peptides was analyzed (Fig. 4A). It was observed that 12- and 15-mer peptides inhibited the reaction of anti-alpha -D-mannopyranoside pAbs with the solid phase 12-mer peptide in a dose-dependent manner, but the 6-mer peptide did not bind to the pAbs in the concentration range of up to 2 mM. Despite the fact that anti-alpha -D-mannopyranoside pAbs reacted with the 12-mer peptide attached to solid support (Fig. 2C), its inhibition by peptide in solution (Fig. 4A) was more in the case of the 15-mer peptide than in the case of the 12-mer peptide. At a 2 mM peptide concentration, about 45% inhibition was achieved by the 15-mer peptide compared with about 11.5% by the 12-mer peptide. This inhibition pattern probably reflects higher binding affinity of these pAbs to the 15-mer peptide than to the 12-mer peptide.


Fig. 4. Antisugar polyclonal Abs bind to the peptide ligands of ConA. A, inhibition of anti-alpha -D-mannopyranoside-BSA pAbs binding to solid phase 12-mer DT by soluble 12-, 15-, and 6-mer peptides in the competitive ELISA. Constant amounts of Abs were incubated with increasing amounts of these peptides, and binding of free Abs to solid phase 12-mer DT was measured in the ELISA. B, circular dichroism data comparing the three peptides, 15-, 12-, and 6-mer (1, 2, and 3, respectively).
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The circular dichroism spectra (Fig. 4B) of the three peptides reflect the profile of binding to anti-alpha -D-mannopyranoside pAbs. All three peptides showed a positive molar ellipticity at about 230 nm, generally attributed to the aromatic residues (19). This was expected, since these peptides are rich in tyrosine and tryptophan residues. The 12- and 15-mer peptides showed two distinct negative peaks, one at 207 nm and another at 212 nm. However, the 6-mer peptide showed a negative peak at 212 nm and a positive peak at 202 nm. Thus, the CD profile of the 6-mer peptide was markedly different from those of the other two peptides.

Rationally Designed Peptide Ligands of ConA

The CD spectra suggested that the 12- and 15-mer peptides are structurally similar to each other. However, the CD spectrum of the 6-mer peptide was very different from those of the other two peptides. This difference was also reflected in the antibody reactivities. Comparison of the sequences of the three peptides indicated that the carboxyl-terminal ASGS sequence is the most common feature in the 12- and 15-mer peptides. In previous studies (13), the sequence ASGS was incorporated to improve the solubilities of the ConA binding peptides. This sequence is absent in the 6-mer peptide. It could, therefore, be inferred that this region of the sequence may be functionally important. A peptide with the sequence MYWYPYASGS (10-mer) was synthesized by incorporating ASGS at the carboxyl terminus of the 6-mer peptide. Fig. 5A shows comparison between the 10- and 6-mer peptides in antisugar pAbs binding. It is clear that the decapeptide inhibits binding of pAbs to the absorbed 12-mer conjugate in a dose-dependent manner. The control peptide (6- mer) shows no such competitive inhibition in the same concentration range. These data suggest that ASGS in the peptide sequence has direct contribution to the binding with pAbs.


Fig. 5. Binding of designed 10-mer peptide (MYWYPYASGS) to antisugar pAbs. A, inhibition of anti-alpha -D-mannopyranoside-BSA pAbs binding to the solid phase 12-mer DT by soluble MYWYPY and MYWYPYASGS in the competitive ELISA. Constant amounts of pAbs were incubated with increasing amounts of MYWYPY or MYWYPYASGS, and binding of free pAbs to solid phase 12-mer DT was measured by ELISA. B, inhibition of ConA binding to solid phase 12-mer DT by soluble MYWYPY and MYWYPYASGS in the competitive lectin binding assay. Biotinylated ConA in a concentration of 4 µg/ml was incubated with increasing amounts of MYWYPY or MYWYPYASGS, and binding of free biotinylated lectin to absorbed 12-mer DT was measured using peroxidase-labeled streptavidin. C, circular dichroism spectra comparing 6- and 10-mer peptides (1 and 2, respectively).
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The observations based on antisugar antibody reactivity seem consistent with the ConA binding activities (Fig. 5B) and CD profiles of the peptides (Fig. 5C). Unlike in the case of the 6-mer peptide, the CD profile of the 10-mer peptide has a negative molar ellipticity at 207 nm. In fact, the CD spectrum of the 10-mer peptide (Fig. 5C) is similar to that of the 12- and 15-mer peptides (Fig. 4B). Thus, addition of ASGS in the hexapeptide appears to have led to a definite structural fold similar to that of the other nonhomologous peptide ligands of ConA. Furthermore, a competitive ConA binding assay was carried out to investigate whether the designed decapeptide can also bind to ConA. The 6-mer peptide was used again as the control. The results indicated that the 10-mer peptide binds to ConA in a dose-dependent manner, but the 6-mer peptide does not (Fig. 5B), corroborating that the sequence ASGS also participates in the alpha -D-mannopyranoside mimicking by the peptide.

The comparison of binding properties of the 12-, 15-, and 10-mer peptides (Figs. 4 and 5), and their sequences suggested that the 15- and 10-mer peptides have comparable antisugar pAbs binding activity. However, the 12-mer peptide showed significantly lower activity against these antibodies. This difference could be correlated with their sequences. There is Phe at position 3 in the 12-mer peptide compared with Trp in the other two peptides. Therefore, an analog of the 12-mer peptide was synthesized by changing Phe3 to Trp, leading to the sequence DVWYPYASGS (12'-mer), and assayed for the reactivity to antisugar pAbs. The substituted peptide showed significantly higher binding to pAbs (Fig. 6A). The ConA binding of the peptide was also analyzed (Fig. 6B). The binding curve of the 12-mer peptide to ConA showed a sharper increase with respect to peptide concentration compared with that of the 12'-mer peptide. However, there is no significant change in the CD spectrum of the 12-mer peptide after mutation (data not shown). The ConA binding of the 12'-mer peptide was similar to that of the 10-mer peptide (Fig. 5B). A nonspecific peptide of comparable size (PTPSPPMSPLRPG) was used as a negative control for antibody binding (Fig. 6A) as well as ConA binding (Fig. 6B), which showed no significant binding in either case.


Fig. 6. Designed point mutation Phe3 right-arrow Trp in the 12-mer peptide enhances its binding to antisugar pAbs. A, inhibition of anti-alpha -D-mannopyranoside-BSA pAbs to solid phase 12-mer DT by soluble 12-mer (DVFYPYPYASGS), 12'-mer (DVWYPYPYASGS), and 13-mer (PTPSPPMSPLRPG) peptides in the competitive ELISA. Constant amounts of pAbs were incubated with increasing amounts of the peptides, and binding of free pAbs to solid phase 12-mer DT was measured by ELISA. B, inhibition of ConA binding to solid phase 12-mer DT by 12-, 12'-, and 13-mer peptides in the competitive lectin binding assay. Biotinylated lectin in a concentration of 4 µg/ml was incubated with increasing amounts of these peptides, and binding of free biotinylated lectin to absorbed 12-mer DT was measured using peroxidase-labeled streptavidin. A nonspecific peptide (PTPSPPMSPLRPG) of comparable size, when used as a control, did not exhibit any binding either to antisugar pAbs or to ConA.
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DISCUSSION

The complementarity of geometry and charge, expressed in terms of the weak noncovalent forces at the interface, is responsible for mediating molecular recognition. This property is common to diverse molecular interactions, which include enzyme-substrate, protein-nucleic acid, and antigen-antibody. Functional equivalence of the chemically dissimilar molecules sharing common surface topology is, therefore, anticipated. Many such equivalences involving proteinaceous molecules and glycan moieties have been identified. Tendamistat, a regulatory protein inhibitor of alpha -amylase, binds to the carbohydrate binding site of the enzyme through a tripeptide epitope (20, 21). It has been suggested that streptococcal group A carbohydrate induces antibodies cross-reacting with cytokeratin peptides, leading to the clinical manifestations of autoimmune responses (22). The analysis of finer details of the mimicry between peptide and carbohydrate ligands of ConA is relevant for defining structural correlations corresponding to such functionally important protein-carbohydrate mimics.

The peptide ligand (12-mer) not only binds to ConA but can also be displaced by methyl alpha -D-mannopyranoside in a dose-dependent manner. However, this alone is not sufficient to suggest that the two are structurally similar. There are many examples in which an organic molecule can bind to a receptor as strongly as the normal ligand without being structurally similar (23). But the anti-12-mer peptide-specific polyclonal antibodies cross-react with alpha -D-mannopyranoside, and the anti-alpha -D-mannopyranoside-specific polyclonal antibodies cross-react with the peptide, suggesting that the peptide and the carbohydrate ligands are topological equivalents. A truly polyclonal antibody population maps the entire surface of the antigen through the distribution of specificities. It essentially represents a topological map of the antigen. Such an antibody population can also provide a measure of similarity of the reciprocally related ligands. As seen in Fig. 3, the antipeptide antibodies distinguish high and low affinity ligands of ConA in a way comparable with the antisugar antibodies. It is known that methyl alpha -D-mannopyranoside binds to ConA more strongly than methyl alpha -D-glucopyranoside. The antipeptide pAbs bind to methyl alpha -D-mannopyranoside four times as efficiently as to methyl alpha -D-glucopyranoside. This implies that the surface similarity of the peptide ligand is more to the methyl alpha -D-mannopyranoside than to methyl alpha -D-glucopyranoside. As expected, both the anti-12-mer peptide and the anti-alpha -D-mannopyranoside pAbs do not recognize alpha -lactose, which is structurally very different from alpha -D-mannopyranoside. Thus, the antibody population not only provides a topological description of a molecular mimic but can also detect the extent of mimicry.

The three peptide ligands have varying cross-reactivities to the antisugar pAbs. The 6-mer peptide is apparently a weak ligand of ConA. Within the concentration range used in the ELISA, no binding of this peptide to the antibodies was detected. However, 12- and 15-mer peptides show binding to these antibodies. The structural properties of the peptides, analyzed by CD spectroscopy, are consistent with these observations. All the peptide analogs exhibited characteristic structural features in their CD spectra. Short peptides containing a sequence motif in which proline is sandwiched between two aromatic residues is known to have definitive structure in solution (24). The feature corresponding to the beta -sheet secondary structure with a negative ellipticity at 212 nm is shared by all three peptides. However, a characteristic polyproline type II feature detected in the 12- and 15-mer peptides corresponding to negative ellipticity at 207 nm is absent in the 6-mer peptide. Thus, the 12- and 15-mer peptides are structurally closer to each other compared with the 6-mer peptide.

The relative differences in binding of different peptides to antisugar pAbs implies that it is possible to delineate the residues responsible for the topological similarity with the sugar. It had been suggested that the consensus sequence YPY is critical for binding to ConA (13, 14). However, the residues away from YPY also appear to participate in binding. The carboxyl-terminal four residues (ASGS) of these peptides significantly contribute to the topological resemblance to the sugar moiety and the ConA binding activity (Fig. 5). A decapeptide designed by incorporating these four residues at the carboxyl terminus of the 6-mer peptide not only shows better binding to antisugar antibodies but has structural similarity with the 12- and 15-mer peptides. In addition, it binds to ConA, unlike the 6-mer peptide. The hydrogen bonds involving hydroxyl groups from the sugar moieties critically contribute to the ConA-carbohydrate interaction (17, 25). The serine side chains in the ASGS sequence can also be attractive substitutes as hydrogen bonding donors in place of sugar hydroxyl groups. The point mutation at an amino-terminal residue Phe3 right-arrow Trp in the 12-mer peptide (called 12'-mer) improves its binding to antisugar pAbs without significantly affecting its structure. Although the binding affinities of the two analogs, 12-mer and 12'-mer, to ConA appears to be comparable, the mutation seems to have brought about a transition in the nature of binding. The 12-mer analog shows a much sharper increase in the ConA binding compared with that of the 12'-mer analog over the same concentration range of the peptide. ConA is known to exhibit structural and thermodynamic diversity in the mode of interaction with different carbohydrate moieties (25-28). The differences in the nature of binding by various peptide analogs may also reflect similar diversity.

The carboxyl-terminal extension consisting of ASGS and the amino-terminal region incorporating WYPY are common in all peptide analogs sharing a strong topological relationship with the carbohydrate moiety. The three peptides, 10-, 12'-, and 15- mer, which share these two sequence motifs exhibit similar ConA binding activities. The anticarbohydrate pAb binding of these peptides is comparatively high. Also, the circular dichroism spectra of these peptides show significant resemblance with each other. However, the region sandwiched between these two motifs is highly diverse in terms of length and the nature of the residues in the three peptides. Whereas the 15-mer peptide has five residues sandwiched between WYPY and ASGS, the 12'-mer peptide has two, and the 10-mer peptide has none. The two residues in the 12'-mer peptide are not related to the corresponding sandwiched sequence in the 15-mer peptide. It may, therefore, be inferred that this part of the molecule does not significantly contribute to either structure, carbohydrate mimicry or the ConA binding activity of the peptide.

Thus, the analysis involving mutual pAb cross-reactivity of a peptide and a carbohydrate ligand has provided a detailed description of the topological similarity between these two essentially dissimilar molecules. The structural features and the amino acid residues of the peptide ligand, critical for carbohydrate mimicry, were identified. A direct correlation was observed between the extent of carbohydrate mimicry and the ConA binding activities of various peptide analogs. The immunological probe involving pAbs used here in analyzing the topological relationship between peptide and carbohydrate ligands of ConA can also be effective in designing peptidomimetic drugs.


FOOTNOTES

*   This work was supported by the Department of Biotechnology, Government of India. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed. Tel.: 91-11-616-7623 (ext. 234); Fax: 91-11-616-2125; E-mail: dinakar{at}nii.ernet.in.
1    The abbreviations used are: ConA, concanavalin A; pAb, polyclonal antibody; BSA, bovine serum albumin; DT, diphtheria toxoid; ELISA, enzyme-linked immunosorbent assay; CD, circular dichroism.

Acknowledgments

We thank Drs. A. Surolia, S. Rath, and J. K. Batra for useful discussions and H. S. Sarna for technical assistance.


REFERENCES

  1. Baum, H., Butler, P., Davies, H., Sternberg, M. J. E., and Burroughs, A. K. (1993) Trends Biochem. Sci. 18, 140-144 [Medline] [Order article via Infotrieve]
  2. Laskowski, M., and Kato, I. (1980) Annu. Rev. Biochem. 49, 593-626 [CrossRef][Medline] [Order article via Infotrieve]
  3. Grewal, N., Talwar, G. P., and Salunke, D. M. (1994) Protein Eng. 7, 205-211 [Abstract]
  4. Moore, G. J. (1994) Trends Phramacol. Sci. 15, 124-129 [CrossRef][Medline] [Order article via Infotrieve]
  5. Beeley, N. (1994) Trends Biotech. 12, 213-216 [Medline] [Order article via Infotrieve]
  6. Gante, J. (1994) Angew. Chem. Int. Ed. Engl. 33, 1699-1720
  7. Liskamp, R. M. J. (1994) Angew. Chem. Int. Ed. Engl. 33, 305-307
  8. Masek, B. B., Merchant, A., and Matthew, J. B. (1993) Proteins Struct. Funct. Genet. 17, 193-202 [Medline] [Order article via Infotrieve]
  9. Nakanishi, H., Ramurthy, S., Raktabutr, A., Shen, R., and Kahn, M. (1993) Gene (Amst.) 137, 51-56 [Medline] [Order article via Infotrieve]
  10. Kuntz, I. D. (1992) Science 257, 1078-1082 [Medline] [Order article via Infotrieve]
  11. Vlatakis, G., Andersson, L. I., Muller, R., and Mosbach, K. (1993) Nature 361, 645-647 [Medline] [Order article via Infotrieve]
  12. Dean, P. M. (ed) (1995) Molecular Similarity in Drug Design, Chapman and Hall, Glasgow, UK
  13. Oldenberg, K. R., Lognathan, D., Goldstein, I. J., Schultz, P. G., and Gallop, M. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5393-5397 [Abstract]
  14. Scott, J. K., Lognathan, D., Easly, R. B., Gong, X., and Goldstein, I. J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5398-5402 [Abstract]
  15. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., and Smith, F. (1956) Anal. Chem. 28, 350-356
  16. Bradford, M. (1976) Anal. Biochem. 72, 248-254 [CrossRef][Medline] [Order article via Infotrieve]
  17. Derewenda, Z., Yariv, J., Helliwell, J. R., Kalb (Gilboa), A. J., Dodson, E. J., Papiz, M. Z., Wan, T., and Campbell, J. (1989) EMBO J. 8, 2189-2193 [Abstract]
  18. So, L. L., and Goldstein, I., J. (1969) Carbohydr. Res. 10, 231-244 [CrossRef]
  19. Chakrabartty, A., Kortemme, T., Padmanabhan, S., and Baldwin, R. L. (1993) Biochemistry 32, 5560-5565 [Medline] [Order article via Infotrieve]
  20. Vertesy, L., Oeding, B., Bender, R., Zepf, K., and Nesemann, G. (1984) Eur. J. Biochem. 141, 505-572 [Abstract]
  21. Hofmann, O., Vertesy, L., and Braunitzer, G. (1985) Biol. Chem. Hoppe-Seyler 366, 1161-1168 [Medline] [Order article via Infotrieve]
  22. Froude, J., Gibofsky, A., Buskirk, D. R., Khanna, A., and Zabriskie, J. B. (1989) Curr. Top. Microbiol. Immunol. 145, 5-26 [Medline] [Order article via Infotrieve]
  23. Wainer, B. H., Wung, W. E., Connors, M., and Rothberg, R. M. (1979) J. Pharmacol. Exp. Ther. 208, 498-506 [Abstract]
  24. Yao, J., Dyson, H. J., and Wright, P. E. (1994) J. Mol. Biol. 243, 754-766 [Medline] [Order article via Infotrieve]
  25. Naismith, J. H., and Field, R. A. (1996) J. Biol. Chem. 271, 972-976 [Abstract/Free Full Text]
  26. Mandal, D. K., Bhattacharyya, L., Koenig, S. H., Brown, R. D., Oscarson, S., and Brewer, C. F. (1994) Biochemistry 33, 1157-1162 [Medline] [Order article via Infotrieve]
  27. Gray, R. D., and Glew, R. H. (1973) J. Biol. Chem. 248, 7547-7551 [Abstract/Free Full Text]
  28. Williams, B. A., Chervenak, M. C., and Toone, E. J. (1992) J. Biol. Chem. 267, 22907-22911 [Abstract/Free Full Text]

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