The neuromuscular junction nicotinic acetylcholine receptor (AChR), a pentameric membrane glycoprotein, is the autoantigen involved in the autoimmune disease myasthenia gravis (MG). In animals immunized with intact AChR and in human MG, the anti-AChR antibody response is polyclonal. However, a small extracellular region of the AChR alpha-subunit, the main immunogenic region (MIR), seems to be a major target for anti-AChR antibodies. A major loop containing overlapping epitopes for several anti-MIR monoclonal antibodies (mAbs) lies within residues alpha 67-76 at the extreme synaptic end of each alpha-subunit: however, anti-MIR mAbs are functionally and structurally quite heterogeneous. Anti-MIR mAbs do not affect channel gating, but are very effective in the passive transfer of MG to animals; in contrast, their Fab or Fv fragments protect the AChR from the pathogenic effects of the intact antibodies. Antibodies against the cytoplasmic region of the AChR can be elicited by immunization with denatured AChR and the precise epitopes of many such mAbs have been identified; however, it is unlikely that such antibodies are present in significant amounts in human MG. Antibodies to other extracellular epitopes on all AChR subunits are present in both experimental and human MG; these include antibodies to the acetylcholine-binding site which affect AChR function in various ways and also induce acute experimental MG. Finally, anti-AChR antibodies cross-reactive with non-AChR antigens exist, suggesting that MG may result from molecular mimicry. Despite extensive studies, many gaps remain in our understanding of the antigenic structure of the AChR; especially in relation to human MG. A thorough understanding of the antigenic structure of the AChR is required for an in-depth understanding, and for possible specific immunotherapy, of MG.

We have investigated the mechanism of functional cooperativity between specificity protein 1 (Sp1) and hepatocyte nuclear factor-4 (HNF-4) on the human apolipoprotein CIII (apoCIII) promoter. Cotransfections in Drosophila SL2 cells that lack endogenous Sp1 or Sp1-related activities showed that HNF-4 and Sp1 synergistically transactivate the -890/+24 apoCIII promoter up to 150-fold. Synergistic transactivation required the HNF-4 binding site of the apoCIII enhancer. Deletion of part of the Ser/Thr-rich and Gln-rich domain or the C-terminal domain of Sp1 decreased, and deletion of residues 501-610 of Sp1 increased, the functional cooperativity between Sp1 and HNF-4. Physical interactions between the two factors were demonstrated by glutathione S-transferase pull-down and co-immunoprecipitation assays. The amino terminal domain of both factors and the carboxy terminal domain of Sp1 contribute to these interactions. Antagonism between HNF-4 and Sp1 was demonstrated on homopolymeric promoters containing multiple binding sites for either factor, suggesting that the synergism between the two factors occurs only when both factors are bound simultaneously to the DNA. The observed physical interactions between Sp1 and HNF-4 in the context of the apoCIII promoter may explain in part their in vitro and in vivo synergism in the transcriptional activation of the apolipoprotein A-I/apoCIII/apolipoprotein A-IV gene cluster.

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The crystal structure of the Fab fragment of a rat monoclonal antibody, number 192, with a very high affinity (K d 0.05 nm) for the main immunogenic region of the human muscle acetylcholine receptor (AChR), has been determined and refined to 2.4 A Ê resolution by X-ray crystallographic methods. The overall structure is similar to a Fab (NC6.8) from a murine antibody, used as a search model in molecular replacement. Structural comparisons with known antibody structures showed that the conformations of the hypervariable regions H1, H2, L1, L2, L3 of Fab192 adopt the canonical structures 1, 1, 2, 1, and 1, respectively. The surface of the antigen-binding site is relatively planar, as expected for an antibody against a large protein antigen, with an accessible area of 2865 A Ê 2 . Analysis of the electrostatic surface potential of the antigen-binding site shows that the bottom of the cleft formed in the center of the site appears to be negatively charged. The structure will be useful in the rational design of very high affinity humanized mutants of Fab192, appropriate for therapeutic approaches of the model autoimmune disease myasthenia gravis.