Antibodies against a peptide of cholera toxin differing in cross-reactivity with the toxin differ in their specific interactions with the peptide as observed by 1H NMR spectroscopy.
The interactions between the aromatic residues of the monoclonal antibody TE34, and its peptide antigen CTP3, have been studied by 2D TRNOE difference spectroscopy. The sequence of CTP3 corresponds to residues 50-64 of the B subunit of cholera toxin (VEVPGSQHIDSQKKA). Unlike two previously studied anti-CTP3 antibodies (TE32 and TE33), the TE34 antibody does not bind the toxin. The off-rate of CTP3 from TE34 was found to be too slow to measure strong TRNOE cross-peaks between the antibody and the peptide. Much faster off-rates, resulting in a strong TRNOE, were obtained for two peptide analogues: (a) CTP3 with an amide in the C-terminus (VEVPGSQHIDSQKKA-NH2) and (b) a truncated version of the peptide (N-acetyl-IDSQKKA). These modifications do not interfere significantly either with the interactions of the unmodified part of the peptide with the antibody or with intramolecular interactions occurring in the epitope recognized by the antibody. The combined use of these peptides allows us to study the interactions between the antibody and the whole peptide. Two tyrosine residues and one or more tryptophan and phenylalanine residues have been found to interact with histidine-8, isoleucine-9, aspartate-10, lysine-13 and/or lysine-14, and alanine-15 of the peptide. In the bound peptide, we observe interactions of a lysine residue with aspartate-10 beta protons. While the peptide epitope recognized by TE34 is between histidine-8 and the negatively charged C-terminus, that recognized by TE32 and TE33 is between residues 3 and 10 of the peptide.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Polymer Research, Weizmann Institute of Science, Rehovot, Israel.
J
J
Anglister
B
B
Zilber
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology*; D000941:Antigens / Q000276:immunology; D002772:Cholera Toxin / Q000276:immunology*; D003429:Cross Reactions; D000939:Epitopes / Q000276:immunology; D008239:Lysine / Q000276:immunology; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology*; D010649:Phenylalanine / Q000276:immunology; D011522:Protons; D014364:Tryptophan / Q000276:immunology; D014443:Tyrosine / Q000276:immunology
D000906:Antibodies; D000941:Antigens; D000939:Epitopes; D010446:Peptide Fragments; D011522:Protons; C064138:N-acetyl-cholera toxin (50-64)-3-amide; D014443:Tyrosine; D010649:Phenylalanine; C050310:cholera toxin, B subunit (50-64); D014364:Tryptophan; D002772:Cholera Toxin; D008239:Lysine