Probing antibody diversity by 2D NMR: comparison of amino acid sequences, predicted structures, and observed antibody-antigen interactions in complexes of two antipeptide antibodies

Abstract

The interactions between the aromatic amino acids of two monoclonal antibodies (TE32 and TE33) with specific amino acid residues of a peptide of cholera toxin (CTP3) have been determined by two-dimensional (2D) transferred NOE difference spectroscopy. Aromatic amino acids are found to play an important role in peptide binding. In both antibodies two tryptophan and two tyrosine residues and one histidine residue interact with the peptide. In TE33 there is an additional phenylalanine residue that also interacts with the peptide. The residues of the CTP3 peptide that have been found to interact with the antibody are val 3, pro 4, gly 5, gln 7, his 8, and asp 10. We have determined the amino acid sequences of the two antibodies by direct mRNA sequencing. Computerized molecular modeling has been used to build detailed all-atom models of both antibodies from the known conformations of other antibodies. These models allow unambiguous assignment of most of the antibody residues that interact with the peptide. A comparison of the amino acid sequences of the two anti-CTP3 antibodies with other antibodies from the same gene family reveals that the majority of the aromatic residues involved in the binding of CTP3 are conserved although these antibodies have different specificities. This similarity suggests that these aromatic residues create a general hydrophobic pocket and that other residues in the complementarity-determining regions (CDRs) modulate the shape and the polarity of the combining site to fit the specific antigens.

Structure and function of cholera toxin and the related Escherichia coli heat-labile enterotoxin.

“…Antipeptide monoclonal antibodies against various segments of CT have been used in several types of studies, including some directed at vaccine development. Peptideantibody interactions were studied by nuclear magnetic resonance methods (7,8,172), which defined contact interactions between antibodies and a highly immunogenic peptide, CTB-3 (143), corresponding to residues CTB:50 through C`TB:64 in the holotoxin. Aromatic amino acids of two of the elicited antibodies (TE32 and TE33) were found to interact with Val, Pro, Gly, Gln, His, and Asp in the peptide, homologous to CTB residues 50, 53, 54, 56, 57, and 58, respectively, all of which are part of an exposed flexible loop near the binding pocket of LT (255).…”

“…Work by Shen et al (245), for example, made use of the receptor specificity of CT to demonstrate that glycolipids inserted into lipid vesicles retained their biological function and orientation, showing by gel ifitration that labeled CT coeluted with a GM1-containing vesicle-rich fraction. Other uses include studies of immune system stimulation (6,114,157), nuclear magnetic resonance or HPLC studies of antibody diversity and antibody-antigen interactions (8,172,259), and studies of vaccine development, in which it is used as a model (216). Hybrid proteins consisting of B subunit and foreign antigens for immunoprotection have generated some recent interest (42).…”

Structure of an anti‐cholera toxin antibody Fab in complex with an epitope‐derived D‐peptide: a case of polyspecific recognition

“…The murine anti-cholera toxin antibody TE33 (IgG1, k light chain) (Anglister et al, 1988;Levy et al, 1989) recognizes a linear epitope with the sequence VEVPGSQHIDSQKKA from the toxin B subunit (Jacob et al, 1983(Jacob et al, , 1984a(Jacob et al, , 1984bShoham, 1993). Subsequently, it was shown that the linear epitope sequence could be reduced to a minimal sequence length of nine residues (VPGSQHIDS), binding with a K D of 1 Â 10 À5 M , that is, about one order of magnitude lower than the original epitope peptide (K D ¼ 8.33 Â 10 À7 M (Anglister et al, 1988)) but still well within the normal range of affinities of antigens to antibodies (10 À3 -10 À10 M (Van Regenmortel, 1998)).…”

A T1 rho-filtered two-dimensional transferred NOE spectrum for studying antibody interactions with peptide antigens

“…The difference between the NOESY spectrum ofthe Fab in the presence of peptide excess and that of the peptide saturated Fab revealed only transferred NOE and exchange cross-peaks. These interactions were assigned to their corresponding protons using specific deuteration of the antibody and a predicted model for its binding site structure (20,21 ). Although the difference method was successful in elucidating the intermolecular interactions between the aromatic amino acids of the three antibodies and the peptide antigen, they were quite cumbersome and required good matching of spectra measured for two different samples.…”