A family of concanavalin A-binding peptides from a hexapeptide epitope library.

Abstract

The lectin concanavalin A (Con A) binds methyl a-D-mannopyranoside (MeaMan) as well as a-Dmannosyl groups at the nonreducing terminus of oligosaccharides. Ligand peptides that mimic the binding of MeaMan to Con A were identified from screening an epitope library composed of rilamentous phage displaying random hexapeptides. A consensus sequence was identified among affinitypurified phage; Con A binds phage bearing this sequence and is inhibited from doing so by MeaMan. When tested for binding against a panel of lectins, phage bearing this sequence bind only weakly to a closely related D-mannose-binding lectin, indicating that binding to Con A is highly selective. A synthetic peptide bearing the consensus sequence blocks the precipitation of Con A by dextran with an inhibition strength equivalent to that of methyl a-D-glucopyranoside. These results demonstrate that the specificity of Con A is not limited to carbohydrates and that highly selective sugar-mimics for lectins of plant, animal, or bacterial origin may be identified from epitope libraries.The lectin Con A comprises a significant fraction of the protein in the jack bean (Canavalia ensiformis). It is a tetramer, consisting of four identical subunits that bind with moderate affinity M) to the a anomers of D-mannose and D-glucose (1). When oligosaccharides containing these sugars are displayed on a cell surface, Con A binds with high avidity as a result of multivalent interactions. Because of this high avidity and its specificity for particular sugars, the function of Con A is presumed to involve binding to D-mannosyl-containing oligosaccharides as part of a specific cell recognition process. In an effort toward developing specific target-ligands for lectins, we have discovered a set of related peptides that bind at or near the sugar-binding site of Con A with affinity equivalent to that of methyl a-Dglucopyranoside (MeaGlc). These peptides were obtained from screening a hexapeptide epitope library with Con A.The epitope library consists of 200 million filamentous phage clones that display about 70o of all possible hexapeptides (2). The use of epitope libraries in identifying ligand peptides has been demonstrated for protein-binding antibodies (2-4), as well as for the biotin-binding site of streptavidin (5). The latter result was the first demonstration that epitope libraries can be used to discover peptides that mimic the binding of nonpeptide ligands.Phage bearing Con A-binding sequences were affinityenriched from the library and identified by (i) exhibiting a consensus sequence that is shared among randomly chosen phage and by their ability (ii) to selectively bind to Con A in ELISA, (iii) to be significantly retained by immobilized Con A, and (iv) to be specifically inhibited from binding and enrichment on Con A by methyl a-D-mannopyranoside (Mea Man).Furthermore, hexapeptide bearing the consensus sequence was shown to bind directly to Con A and to inhibit the precipitation of Con A by dextran with a strength equivalent to that of MeaGlc, where...

Protein ligands of the human adenovirus type 2 outer capsid identified by biopanning of a phage-displayed peptide library on separate domains of wild-type and mutant penton capsomers.

“…For this purpose we used the technique of affinity selection of complementary peptide ligands from a filamentous bacteriophage-displayed random peptide library on immobilized ligates (Parmley and Smith, 1988;Cwirla et al, 1990;Scott and Smith, 1990). This technique has been successfully applied to the identification of (i) the epitope recognized by a monoclonal antibody (Stephen and Lane, 1992;Yayon et al, 1993), (ii) the immunofootprints of pathogens in human sera (Folgori et al, 1994) and (iii) complementary peptide sequences in partner proteins of integrins (Koivunen et al, 1993(Koivunen et al, , 1994, cellular chaperones (Blond-Elguindi et al, 1993) or concanavalin A (Oldenburg et al, 1992;Scott et al, 1992).…”

High-affinity urokinase receptor antagonists identified with bacteriophage peptide display.

“…Bacteriophage display permits the expression of millions of peptides (14,15) or proteins (16)(17)(18) on the surface of bacteriophage particles, biochemical selection of ligands, and identification by DNA sequencing of the packaged bacteriophage genomes. Peptide ligands for several soluble proteins including streptavidin, concanavalin A, integrins, such as gpllb,, and a variety of antibodies have been identified (15,(19)(20)(21)(22) as well as antibody fiagments that bind to cells (23). We report here the identification and characterization of peptide antagonists with nanomolar affinity for the human uPAR by using a 15-mer peptide library.…”

Structural Evaluation of a Mimicry-Recognizing Paratope: Plasticity in Antigen–Antibody Interactions Manifests in Molecular Mimicry

“…Methyl a-D-mannopyranoside and a dodecapeptide (DVFYPY-PYASGS) known to bind Con A with comparable affinities have been studied extensively as a model system to address molecular mimicry in the humoral immune response (17)(18)(19). Polyclonal sera generated against methyl a-D-mannopyranoside cross-recognize the dodecapeptide, and correspondingly antipeptide sera cross-react with the mannopyranoside (20).…”