The N-terminal extracellular domain (amino acids 1-210; h␣-(1-210)) of the ␣ subunit of the human muscle nicotinic acetylcholine receptor (AChR), bearing the binding sites for cholinergic ligands and the main immunogenic region, the major target for anti-AChR antibodies in patients with myasthenia gravis, was expressed in the yeast, Pichia pastoris. The recombinant protein was water-soluble and glycosylated, and fast protein liquid chromatography analysis showed it to be a monomer. h␣-(1-210) bound 125 I-␣-bungarotoxin with a high affinity (K d ‫؍‬ 5.1 ؎ 2.4 nM), and this binding was blocked by unlabeled d-tubocurarine and gallamine (K i ϳ7.5 mM). Interestingly, 125 I-␣-bungarotoxin binding was markedly impaired by in vitro deglycosylation of h␣-(1-210). Several monoclonal antibodies that show partial or strict conformation-dependent binding to the AChR were able to bind to h␣-(1-210), as did antibodies from a large proportion of myasthenic patients. These results suggest that the extracellular domain of the human AChR ␣ subunit expressed in P. pastoris has an apparently near native conformation. The correct folding of the recombinant protein, together with its relatively high expression yield, makes it suitable for structural studies on the nicotinic acetylcholine receptor and for use as an autoantigen in myasthenia gravis studies. The nicotinic acetylcholine receptor (AChR)1 at the neuromuscular junction is a member of the superfamily of ligandgated ion channels that also includes the glycine, ␥-aminobutiric acid A, and 5-HT 3 receptors (1). The AChR is a transmembrane glycoprotein (M r ϳ290000) consisting of five homologous subunits in the stoichiometry ␣ 2 ␤␥␦ (embryonic) or ␣ 2 ␤⑀␦ (adult). Each subunit consists of an N-terminal extracellular domain (ϳ210 residues) followed by three transmembrane domains, a large cytoplasmic loop, a fourth transmembrane domain, and a short, extracellular C-terminal tail (2, 3). The N-terminal extracellular domain of the ␣ chain (␣-(1-210)) contains both the binding sites for cholinergic ligands (4) and the MIR, the major target for autoantibodies in both MG and experimental models of MG (5-7). The major loop of the overlapping epitopes for several anti-MIR monoclonal antibodies (mAbs) has been localized between residues 67 and 76 of the ␣ subunit (8, 9). Previous experiments have shown that the binding sites for both acetylcholine and ␣-BTX are located close to two adjacent cysteine residues at positions 192 and 193 of the ␣ subunit (10). Moreover, other distinct regions on either the ␣ subunit (11, 12) or the adjacent ␥ or ␦ subunits (13) have been shown to contribute to ␣-BTX binding, whereas the role of glycosylation at residue ␣141 (14 -16) requires further study.These unique characteristics of the ␣ subunit have led to its being extensively studied in several laboratories. The expression of full-length Torpedo (15-17) or mouse (18) AChR ␣ subunits in heterologous protein expression systems has shown that the ␣ subunit, independently of other subunits, acquires a mature...

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The hepatitis C virus internal ribosome entry site (IRES) binds directly to the 40S ribosomal subunit via domains III/IV while domain II induces conformational changes on the ribosome which have been implicated in the decoding process. Here, we performed an extensive mutational study within the apical portion of domain II in order to address the functional role of this region on translation. Our results showed that the conservation of most nucleotides in this region was only partially related to the IRES function. Notwithstanding, however, selected single point mutations within the apical loop had a deleterious effect on IRES activity. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Antibodies against muscle acetylcholine receptor (AChR) undoubtedly play a critical role in the pathology of most myasthenia gravis (MG) cases. Selective elimination of the majority of these antibodies should result in a considerable improvement of the MG symptoms. Such a specific elimination could be achieved by AChR-based immunoadsorbents. However, sufficient quantities of native human AChR are not available while bacterially expressed recombinant domains of the AChR are unable to bind satisfactorily MG antibodies. We have undertaken the production of the extracellular domains of human AChR subunits in eukaryotic systems, in native-like conformation, for their use as potent immunoadsorbents. The N-terminal extracellular domain (amino acids 1-210; alpha(1-210)) of the alpha(1) subunit of the human muscle AChR was expressed in the yeast Pichia pastoris. The polypeptide was water-soluble, glycosylated, and in monomer form. The alpha(1-210) bound 125I-alpha-bungarotoxin (125I-alpha-BTX) with a high affinity (Kd = 5.1 +/- 2.4 nM), and this binding was blocked by unlabeled d-tubocurarine and gallamine. Several conformation-dependent anti-AChR antibodies were able to bind alpha(1-210) as did antibodies from a large proportion of MG patients. The purified protein was subsequently immobilized on Sepharose-CNBr and was used to immunoadsorb anti-AChR antibodies from 64 MG sera. It eliminated more than 50% (50-94%) of the anti-AChR antibodies in 20% of the sera, whereas from another 30% of the sera it eliminated 20-60% of their anti-AChR antibodies. Work is in progress for the expression of the extracellular domain of all other muscle AChR subunits. It is expected that their combined use may eliminate the great majority of the anti-AChR antibodies from most MG patients.