NMR study of the complexes between a synthetic peptide derived from the B subunit of cholera toxin and three monoclonal antibodies against it

Abstract

The contact interactions between a synthetic peptide and three different anti-peptide monoclonal antibodies have been studied by nuclear magnetic resonance (NMR). The synthetic peptide is CTP3 (residues 50-64 of the B subunit of cholera toxin) suggested as a possible epitope for synthetic vaccine against cholera. The hybridoma cell lines TE33 and TE32 derived after immunization with CTP3 produce antibodies cross-reactive with the native toxin. The cell line TE34 produces anti-CTP3 antibodies that do not bind the toxin. Selective deuteriation of the antibodies has been used to simplify the proton NMR spectra and to assign resonances to specific types of amino acids. The difference spectra between the proton NMR spectrum of the peptide-Fab complex and that of Fab indicate that the combining site structures of TE32 and TE33 are very similar but differ considerably from the combining site structure of TE34. By magnetization transfer experiments with selectively deuteriated Fab fragment of the antibody, we have found that in TE32 and TE33 the histidine residue of the peptide is buried in a hydrophobic pocket of the antibody combining site, formed by a tryptophan and two tyrosine residues. The hydrophobic nature of the pocket is further demonstrated by the lack of any pH titration effect on the chemical shift of the C4H of the bound peptide histidine. In contrast, for TE34 we have found only one tyrosine residue in contact with the histidine of the peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural diversity in a conserved cholera toxin epitope involved in ganglioside binding

“…The antipeptide-antibody TE33 is cross-reactive with intact CT, but the binding constant for the intact toxin is a thousandfold lower (Anglister et al, 1988). Similar decrease in the affinity for the native proteins was observed for other antipeptide antibodies cross-reactive with the cognate protein (Berzofsky, 1985).…”

“…This is the case for TE34, another anti-CTP3 antibody that recognizes the carboxy-terminus of the peptide. TE34 does not bind CT at all, even in a solid-phase assay (Anglister et al, 1988). However, this possibility can be ruled out for TE33 because the termini are not involved in binding to the antibody.…”

“…Immunization of laboratory animals with a peptide, CTP3, corresponding to the sequence of residues 50-64, has been shown to cause partial neutralization of CT and LT activities in vitro (Jacob et al, 1983(Jacob et al, , 1984a(Jacob et al, , 1984b. A monoclonal antibody, TE33, raised against the same CTP3 peptide, is cross-reactive with cholera toxin in a solid-phase immunosorbent assay (ELISA) (Anglister et al, 1988), but in solution the affinity of the antibody for the toxin is a thousandfold lower than for CTP3 (Scherf et al, 1992).…”

Folded conformations of antigenic peptides from riboflavin carrier protein in aqueous hexafluoroacetone

“…The other representative examples that suggest a well-ordered conformations of the antigenic sequences are a peptide from antigenic domain of Herpes simplex virus glucoprotein D-1 (Williamson et al, 1986). peptides from torpedo acetylcholine receptor (Chung et al, 1989(Chung et al, , 1991, peptide from B-subunit of cholera toxin (Anglister et al, 1988;Zilber et al, 1990), the principle neutralizing determinant of HIV virus (Zvi et al, 1992), and peptide antigens from the receptor binding domain of Pseudomonas aeruginosa (McInnes et al, 1993).…”

“…Seminal studies from different groups have subsequently established well-ordered conformations for many such antigenic peptides in solution (Dyson et al, 1985(Dyson et al, , 1988(Dyson et al, , 1990McInnes et al, 1993). A structured backbone conformation has also been demonstrated by NMR and crystallographic studies of peptides bound to monoclonal Fab fragments (Anglister et al, 1988;Stanfield et al, 1990;Jin et al, 1992;Scherf et al, 1992;Tsang et al, 1992). The incorporation of specific stereochemical constraints into antigenic peptides that stabilize "native like" conformational states appears to be a viable tool for enhancing immunological characteristics (Kaumaya et al, 1990(Kaumaya et al, , 1992Tuchscherer et al, 1992;Gurunath et al, 1995).…”