The crystal structure of the Fab fragment of a rat monoclonal antibody against the main immunogenic region of the human muscle acetylcholine receptor

The target of most of the autoantibodies against the acetylcholine receptor (AChR) in myasthenic sera is the main immunogenic region (MIR) on the extracellular side of the AChR alpha-subunit. Binding of anti-MIR monoclonal antibodies (mAbs) has been recently localized between residues alpha 67 and alpha 76 of Torpedo californica electric organ (WNPADYGGIK) and human muscle (WNPDDYGGVK) AChR. In order to evaluate the contribution of each residue to the antigenicity of the MIR, we synthesized peptides corresponding to residues alpha 67-76 from Torpedo and human AChRs, together with 13 peptide analogues. Nine of these analogues had one residue of the Torpedo decapeptide replaced by L-alanine, three had a structure which was intermediate between those of the Torpedo and human alpha 67-76 decapeptides, and one had D-alanine in position 73. Binding studies employing six anti-MIR mAbs and all 15 peptides revealed that some residues (Asn68 and Asp71) are indispensable for binding by all mAbs tested, whereas others are important only for binding by some mAbs. Antibody binding was mainly restricted to residues alpha 68-74, the most critical sequence being alpha 68-71. Fish electric organ and human MIR form two distinct groups of strongly overlapping epitopes. Some peptide analogues enhanced mAb binding compared with Torpedo and human peptides, suggesting that the construction of a very antigenic MIR is feasible.

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Location of subunits within the acetylcholine receptor by electron image analysis of tubular crystals from Torpedo marmorata.

The Journal of Cell Biology ◽

10.1083/jcb.105.1.9 ◽

1987 ◽

Vol 105 (1) ◽

pp. 9-18 ◽

Cited By ~ 130

Author(s):

E Kubalek ◽

S Ralston ◽

J Lindstrom ◽

N Unwin

Keyword(s):

Monoclonal Antibody ◽

Binding Sites ◽

Alpha Subunit ◽

Torpedo Marmorata ◽

Fab Fragment ◽

Alpha Subunits ◽

Immunogenic Region ◽

Electron Image ◽

Alpha Beta ◽

Tubular Crystals

The binding sites on the nicotinic acetylcholine receptor of labels specific for the alpha-, beta-, and delta-subunits were determined by electron image analysis, using tubular crystals of receptors grown from the postsynaptic membranes of Torpedo marmorata electric organ. The labels were alpha-bungarotoxin (which attaches to the acetylcholine binding sites on the pair of alpha-subunits), Fab35 (a monoclonal antibody Fab fragment directed against the main immunogenic region of the alpha-subunit), Fab111 (a monoclonal antibody Fab fragment directed against a cytoplasmic site on the beta-subunit), and wheat germ agglutinin (which binds to N-acetylglucosamine residues on the delta-subunit). These labels, bound to receptors in the crystals, were located by comparing labeled with native structures, averaged in each case over more than 5,000 molecules. From the assignments made, we find that the clockwise arrangement of subunits around the receptor, viewed from the synaptic face, is: alpha, beta, alpha, gamma, and delta; that the main immunogenic region is at (or close to) the side of the alpha-subunit; and that the two acetylcholine binding sites are at the synaptic end of the alpha-subunits, 27-28 A from the central axis and approximately 53 A apart. In the crystal lattice, neighboring molecules are paired so that their delta- and alpha-subunits are juxtaposed, an organization that appears to relate closely to the grouping of receptors in vivo.

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