Amphipathic segment of the nicotinic receptor alpha subunit contains epitopes recognized by T lymphocytes in myasthenia gravis.

Autoimmune helper T lymphocytes were selected from the blood of two myasthenic patients of different HLA-DR type, using acetylcholine receptor (AChR) from Torpedo californica. These polyclonal T cell lines were tested for reactivity with three synthetic peptides corresponding to the NH2-terminal region of the human AChR alpha subunit. This segment is a good candidate for T cell epitopes since it has a propensity to form an amphipathic alpha helix. The peptides elicited 10-30% of the response induced by native Torpedo AChR. Different peptides were recognized by the autoreactive T cells of the two patients. These results suggest that the NH2-terminal region of the AChR alpha chain contains T cell-stimulating epitopes, and that the T cell autoimmune response in myasthenia gravis, like the B cell response, is heterogeneous.

Localization of the main immunogenic region of human muscle acetylcholine receptor to residues 67-76 of the alpha subunit.

The majority of antibodies to the acetylcholine receptor (AcChoR), both in the human disease myasthenia gravis and in its experimental models, are directed against an extracellular area of the AcChoR alpha subunit called the main immunogenic region (MIR). We have studied the binding of anti-AcChoR monoclonal antibodies (mAbs) to 26 synthetic peptides corresponding to the hydrophilic parts of the human AcChoR alpha subunit. The binding sites for eight anti-MIR mAbs and for eight anti-alpha-subunit, non-anti-MIR mAbs were localized. Anti-MIR mAbs bound to one peptide corresponding to residues 63-80 of the human alpha subunit. A second panel of peptides corresponding to the various parts of the alpha-subunit segment 63-80 was synthesized. Anti-MIR antibodies bound to a peptide that contained the alpha-subunit sequence 67-76. Thus, a main constituent loop of the MIR is localized between residues 67 and 76 of the alpha subunit.

Main immunogenic region of Torpedo electroplax and human muscle acetylcholine receptor: localization and microheterogeneity revealed by the use of synthetic peptides.

Most anti-nicotinic acetylcholine receptor (AChR) antibodies in myasthenia gravis are directed against an immunodominant epitope or epitopes [main immunogenic region (MIR)] on the AChR alpha-subunit. Thirty-two synthetic peptides, corresponding to the complete Torpedo alpha-subunit sequence and to a segment of human muscle alpha-subunit, were used to map the epitopes for 11 monoclonal antibodies (mAbs) directed against the Torpedo and/or the human MIR and for a panel of anti-AChR mAbs directed against epitopes on the alpha-subunit other than the MIR. A main constituent loop of the MIR was localized within residues alpha 67-76. Residues 70 and 75, which are different in the Torpedo and human alpha-subunits, seem to be crucial in determining the binding profile for several mAbs whose binding to the peptides correlated very well with their binding pattern to native Torpedo and human AChRs. This strongly supports the identification of the peptide loop alpha 67-76 as the actual location of the MIR on the intact AChR molecule. Residues 75 and 76 were necessary for binding of some mAbs and irrelevant for others, in agreement with earlier suggestions that the MIR comprises overlapping epitopes. Structural predictions for the sequence segment alpha 67-76 indicate that this segment has a relatively high segmental mobility and a very strong turning potential centered around residues 68-71. The most stable structure predicted for this segment, in both the Torpedo and human alpha-subunits, is a hairpin loop, whose apex is a type I beta-turn and whose arms are beta-strands. This loop is highly hydrophilic, and its apex is negatively charged. All these structural properties have been proposed as characteristic of antibody binding sites. We also localized the epitopes for mAbs against non-MIR regions. Among these, the epitope for a monoclonal antibody (mAb 13) that noncompetitively inhibits channel function was localized within residues alpha 331-351.