The main immunogenic region of the nicotinic acetylcholine receptor. Identification of amino acid residues interacting with different antibodies.
In myasthenia gravis a highly conserved area of the nicotinic receptor (AcChR) dominates the autoantibody response (main immunogenic region, MIR), and it is formed by residues within the sequence segment 67-76 of the AcChR alpha-subunit. We have studied the binding of eight anti-MIR mAb to synthetic peptides containing the sequence segment 67-76 of the human alpha-subunit, and peptide analogues containing single residue substitutions of this sequence. We used also a peptide where both Asp70 and Asp71 were substituted by glycine residues. The binding of six anti-MIR mAb was strongly influenced by several substitutions. All these mAb required residues Asn68, and Pro69 for binding. Five of them required also Asp71 and Tyr72. Substitution of Asp70, which is an Ala residue in Torpedo AcChR, was irrelevant for the binding of an anti-Torpedo and an anti-Electrophorus mAb, and moderately reduced the binding of an anti-human mAb (no. 203). Substitution of Trp67 moderately reduced the binding of some of these mAbs. A mAb of this group (the antihuman mAb no. 198) bound in a manner only slightly influenced by ionic strength, whereas the binding of the other five mAb of this group was very sensitive to the ionic strength. Two anti-Electrophorus MIR mAb bound similarly to all peptide analogues in low ionic strength. At high ionic strength only the peptide analogue where Asp 70 was changed to a Gly residue bound significantly. This may indicate that the Electrophorus MIR has an uncharged residue at this position, as does Torpedo AcChR. Residues at position 73, 74, 75, and 76 were of little or no importance for the binding of all anti-MIR mAb. A free amino terminus was essential for the binding of most mAb. The results of competition experiments between different peptides and native AcChR for mAb binding were consistent with those obtained in direct binding experiments.
Bellone
M
M
Department of Biochemistry, University of Minnesota, St. Paul 55108.
Tang
F
F
Milius
R
R
Conti-Tronconi
B M
BM
D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D004593:Electrophorus; D006801:Humans; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000276:immunology / Q000378:metabolism; D009994:Osmolar Concentration; D011863:Radioimmunoassay; D011978:Receptors, Nicotinic / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D014101:Torpedo
D000911:Antibodies, Monoclonal; D009842:Oligopeptides; D011978:Receptors, Nicotinic