Kathryn E. McLane

The Nicotinic Acetylcholine Receptor: Structure and Autoimmune Pathology

The nicotinic acetylcholine receptors (AChR) are presently the best-characterized neurotransmitter receptors. They are pentamers of homologous or identical subunits, symmetrically arranged to form a transmembrane cation channel. The AChR subunits form a family of homologous proteins, derived from a common ancestor. An autoimmune response to muscle AChR causes the disease myasthenia gravis. This review summarizes recent developments in the understanding of the AChR structure and its molecular recognition by the immune system in myasthenia.

Epitope mapping of polyclonal and monoclonal antibodies against two α-bungarotoxin-binding α subunits from neuronal nicotinic receptors

Abstract Recently, cDNAs for α subunits of two different neuronal α-bungarotoxin-binding proteins (αBgtBP) were isolated from chick brain, designated αBgtBP α1 and αBgtBP α2. These are now also referred to as subunits α7 and α8, respectively. Expression studies in Xenopus oocytes have indicated that α7 subunits are able to form cation channels that are sensitive to nicotinic ligands, and therefore represent bona fide nicotinic acetylcholine receptor subunits. Polyclonal and monoclonal antibodies (mAbs) have been produced against: (i) affinity-purified chick brain αBgtBP; and (ii) fusion proteins containing the unique cytoplasmic sequences α7(327–412) and α8(293–435). Here, synthetic overlapping peptides corresponding to their deduced amino acid sequence are used to map the epitopes recognized by the different antibodies. The polyclonal response to affinity-purified αBgtBPs and the fusion proteins indicates that sequence segments 290–420 of both subunits contain several major and minor epitopes. mAbs selected for their ability to bind both native and denatured αBgtBPs isolated from chick brain also recognise subunit-specific sequential epitopes within the sequence segment 290–420. The epitopes recognized by the mAbs correspond to the minor epitopes defined using antisera. The mAbss characterized in these studies will provide useful probes for further studies of αBgtBP structure and histological localization.

Amino acid residues forming the interface of a neuronal nicotinic acetylcholine receptor with κ-bungarotoxin: A study using single residue substituted peptide analogs

kappa-Bungarotoxin is a high affinity antagonist of neuronal nicotinic acetylcholine receptors of the alpha 3 subtype. Three sequence segments of the alpha 3 subunit that contribute to forming the binding site for kappa-bungarotoxin were previously located using synthetic peptides corresponding to the complete alpha 3 subunit, i.e., alpha 3(1-18), alpha 3(50-71) and alpha 3(180-201). Here we use single residue substituted peptide analogs of the alpha 3(50-71) sequence, in which amino acids are sequentially replaced by Gly, to determine which residues are important for kappa-bungarotoxin binding activity. Although no single substitution obliterated kappa-bungarotoxin binding, several amino acid substitutions lowered the affinity for kappa-bungarotoxin--i.e., two negatively charged residues (Glu51 and Asp62), and several aliphatic and aromatic residues (Leu54, Leu56, and Tyr63). These results indicate that the interface of the alpha 3 subunit with kappa-bungarotoxin involves primarily hydrophobic interactions, and a few negatively charged residues.

Species- and Subtype-Specific Recognition by Antibody WF6 of a Sequence Segment Forming an α-Bungarotoxin Binding Site on the Nicotinic Acetylcholine Receptor α Subunit

AbstractThe monoclonal antibody WF6 competes with acetylcholine and α-bungarotoxin (α-BGT) for binding to the Torpedo nicotinic acetylcholine receptor (nAChR) α1 subunit. Using synthetic peptides corresponding to the complete Torpedo nAChR α1 subunit, we previously mapped a continuous epitope recognized by WF6, and the prototope for α-BGT, to the sequence segment α1(181–200). Single amino acid substitution analogs have been used as an initial approach to determine the critical amino acids for WF6 and α-BGT binding. In the present study, we continue our analysis of the structural features of the WF6 epitope by comparing its cross-reactivity with synthetic peptides corresponding to the α1 subunits from the muscle nAChRs of different species, the rat brain α2, α3, α4 and α5 nAChR subtypes, and the chick brain α-BGT binding protein subunits, αBGTBP α1 and αBGTBP α2. Our results indicate that WF6 is able to cross-react with the muscle α1 subunits of different species by virtue of conservation of several critic...

Formation of mannosyl-containing lipid-linked sugars by membranes fromPenicillium charlesii

Abstract A membrane fraction from Penicillium charlesii was shown to catalyze the transfer of mannosyl residues from GDP- d -mannose to dolichyl-phosphate. Based upon the concentration of eluent required to elute the products from DEAE(acetate) resin compared with the authentic dolichyl-phospho-mannose, it was found that the system formed dolichyl-phospho-mannose and dolichyl-pyrophosphomannose-containing oligosaccharides. Synthesis of dolichyl-phospho-mannose required the addition of both dolichyl-phosphate and MgCl 2 to the reaction mixture as well as the membrane preparation. With MnCl 2 as the added cofactor, a trisaccharide was the predominant mannose-containing saccharide formed; lesser quantities of tetra- and larger saccharides were formed. The data suggest that MnCl 2 served as cofactor in the synthesis of both dolichyl-phospho-mannose and dolichyl-phospho-(mannose) x . Mannose incorporation from GDP- d -mannose into the two classes of lipid-linked sugars was not inhibited by tunicamycin which suggests that the mannose-containing oligosaccharide is without N -acetyl- d -glucosamine residues. In contrast, bacitracin or amphomycin added to the reaction mixture inhibited formation of dolichyl-phospho-mannose and to a lesser extent dolichyl-pyrophospho-(mannose) x .