Antigenic modulation of human myotube acetylcholine receptor by myasthenic sera. Serum titer determines receptor internalization rate.

Antibodies to the acetylcholine receptor (AChR) added to AChR-bearing muscle cells cross-link the receptors, thus increasing their internalization and degradation rate (antigenic modulation). This mechanism contributes to AChR loss in myasthenia gravis. Until recently, antigenic modulation has been studied in animal tissues, where only a small fraction of human anti-AChR antibodies bind.

Localisation of the main immunogenic region of the nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor from Torpedo marmorata was digested using papain and the reaction products separated by SDS gel electrophoresis and characterised by immunoblotting using labelled alpha-bungarotoxin, polyclonal antibodies to synthetic peptides and monoclonal antibodies to the main immunogenic region (MIR). Using this approach, it was possible to show that the MIR is located N-terminal to all or part of peptide 151-169 (peptide P1) of the alpha-chain and that papain cleaves the alpha-chain between Asn 141 and peptide P1.

Decrease in acetylcholine-receptor content of human myotube cultures mediated by monoclonal antibodies to alpha, beta and gamma subunits.

One of the two main causes of acetylcholine-receptor loss in myasthenia gravis is antigenic modulation, i.e. accelerated internalization and degradation rate by antibody-crosslinking.

Characteristics of monoclonal antibodies to denatured Torpedo and to native calf acetylcholine receptors: species, subunit and region specificity.

Seventy-five monoclonal antibodies (mAbs) to sodium dodecyl sulfate-denatured Torpedo californica (66 mAbs) and intact fetal calf (9 mAbs) acetylcholine receptor (AChR) were produced. These mAbs were characterized for subunit, region and species specificity, for Ig class and subclass, for protein A binding and for antigen-crosslinking capacity. Fourteen were identified as anti-alpha, 35 were anti-beta, 8 were anti-gamma and 15 were anti-delta.

Role of the main immunogenic region of acetylcholine receptor in myasthenia gravis. An Fab monoclonal antibody protects against antigenic modulation by human sera.

Antigenic modulation of acetylcholine receptor (AChR), i.e., acceleration of its internalization and degradation rate by antibody-cross-linking, is considered to be one of the two main causes of AChR loss in myasthenia gravis (MG).

Lipid-dependent recovery of alpha-bungarotoxin and monoclonal antibody binding to the purified alpha-subunit from Torpedo marmorata acetylcholine receptor. Enhancement by noncompetitive channel blockers.

Recently the purified alpha-subunit from Torpedo marmorata acetylcholine receptor was shown to bind alpha-bungarotoxin with a KD approximately 3 nM in the presence of sodium dodecyl sulfate (Tzartos, S.J., and Changeux, J.

Spectrotypic analysis of antibodies to acetylcholine receptors in experimental autoimmune myasthenia gravis.

We have studied the isoelectric focusing pattern of antibodies expressed in rats with experimental autoimmune myasthenia gravis (EAMG) induced by immunization with acetylcholine receptors (AChR) purified from Torpedo californica. Sera or tissue eluates were obtained at intervals in the course of disease and subjected to isoelectric focusing. Subsequently, the focused antibodies were detected by autoradiography of gels labelled with 125I-alpha-bungarotoxin conjugated AChR.

Antigenic sites of the nicotinic acetylcholine receptor cannot be predicted from the hydrophilicity profile.

The amino acid sequences of the polypeptide chains of the acetylcholine receptor have recently been published. From the hydrophilicity profiles, it has been proposed that residues 161-166 of the alpha-chain might be an important antigenic site. We have synthesised a peptide containing this sequence and raised antisera to it.

Structure and transmembrane nature of the acetylcholine receptor in amphibian skeletal muscle as revealed by cross-reacting monoclonal antibodies.

A collection of 126 monoclonal antibodies (mAbs) made against acetylcholine receptors (AChRs) from the electric organs of Torpedo californica or Electrophorus electricus was tested for cross-reactivity with AChRs in cryostat sections of skeletal muscle from Rana pipiens and Xenopus laevis by indirect immunofluorescence. 49 mAbs (39%) cross-reacted with AChRs from Rana, and 25 mAbs (20%) cross-reacted with AChRs from Xenopus. mAbs specific for each of the four subunits of electric organ AChR (alpha, beta, gamma, delta) cross-reacted with AChRs from each amphibian species.

Comparison of embryonic and adult torpedo acetylcholine receptor by sedimentation characteristics and antigenicity.

The acetylcholine receptor (AChR) from Torpedo marmorata electric organ exists in a light form (α2βγδ) of apparent molecular weight 250,000. The association of two light forms via an intermolecular δ-δ disulfide bridge results in the AChR heavy form. In adult Torpedo electric organ extracts the heavy form constitutes about 70% of the AChR.

Transmembrane orientation of an early biosynthetic form of acetylcholine receptor delta subunit determined by proteolytic dissection in conjunction with monoclonal antibodies.

The transmembrane topology of acetylcholine receptor (AChR) delta subunit, synthesized in vitro and co-translationally integrated into dog pancreas rough microsomal membranes, was studied using limited proteolysis and domain-specific immunoprecipitation. Forty-four kilodaltons (kd) of the 65-kd delta subunit comprise a single fragment that is inaccessible to exhaustive proteolytic digestion from the cytoplasmic surface of the membrane by trypsin, chymotrypsin, thermolysin, and pronase. Previously, we have shown that this 44-kd "protected" fragment contains the amino terminus of the intact molecule and all of the core oligosaccharides (Anderson, D.

Demonstration of a main immunogenic region on acetylcholine receptors from human muscle using monoclonal antibodies to human receptor.

Eleven cloned hybridomas which secrete antibodies to acetylcholine receptors from human muscle have been prepared. All of these monoclonal antibodies to have the same basic specificity as shown by competition for binding to the main immunogenic region on the receptor, but these antibodies differ in fine specificity as shown by reaction with denatured receptor subunits and interspecies cross-reaction.

Immunohistochemical localization of monoclonal antibodies to the nicotinic acetylcholine receptor in chick midbrain.

We used the indirect immunofluorescence method to determine the crossreactivity of a library of 57 monoclonal antibodies (mAbs) against each of the subunits of the nicotinic acetylcholine receptor (nAcChoR) isolated from Torpedo and Electrophorus electric organs or from fetal calf and human muscle, with specific neural elements in the midbrain of the chick. Out of 17 mAbs that recognized motor end plates on chick muscle, 14 produced a similar pattern of labeling in the midbrain: the neuronal perikarya and dendrites in the lateral spiriform nucleus (SpL) were intensely labeled, and there was moderate labeling of fibers in certain of the deeper layers of the optic tectum, which disappeared after the SpL was destroyed electrolytically. Two lines of evidence suggest that the mAbs may be crossreacting with nAcChoRs in the midbrain.

Characterization of the mRNA for mouse muscle acetylcholine receptor alpha subunit by quantitative translation in vitro.

The nicotinic acetylcholine receptor of mammalian skeletal muscle is a multisubunit membrane glycoprotein whose synthesis is regulated by developmental and physiological cues. We report here the identification and characterization of the primary translation product of alpha subunit mRNA. The alpha subunit synthesized in rabbit reticulocyte lysate is approximately 2000 larger in apparent molecular weight than the native alpha subunit polypeptide found in acetylcholine receptor.

High affinity binding of alpha-bungarotoxin to the purified alpha-subunit and to its 27,000-dalton proteolytic peptide from Torpedo marmorata acetylcholine receptor. Requirement for sodium dodecyl sulfate.

Intact nicotinic acetylcholine receptor (AChR) tightly binds alpha-bungarotoxin. The two toxin-binding sites are presumed to be on the two alpha-subunits, either on or near the ACh-binding sites. Isolated alpha-subunits have been found to maintain weak binding to alpha-bungarotoxin (KD approximately 0.

Inhibition of glycosylation with tunicamycin blocks assembly of newly synthesized acetylcholine receptor subunits in muscle cells.

We have characterized the oligosaccharide chains of the alpha subunit of acetylcholine receptor of the clonal mouse muscle cell line BC3H-1 by their sensitivity to end-beta-N-acetylglucosaminidase H and by comparison of the native glycosylated polypeptide with the nonglycosylated form made in tunicamycin-treated cells. These studies indicate that the native alpha subunit has a single N-asparagine-linked oligosaccharide chain of the "high mannose" or "simple" type. Furthermore, these results considered in light of our previous characterization of the alpha subunit synthesized in vitro suggest that the alpha subunit contains no "complex"-type N-linked oligosaccharide chains.

Specificities of antibodies to acetylcholine receptors in sera from myasthenia gravis patients measured by monoclonal antibodies.

The pattern of antibody specificities in sera from patients with myasthenia gravis (MG) was determined by the ability of monoclonal antibodies against defined determinants on the acetylcholine receptor molecule to inhibit binding of the serum antibodies to receptor from human muscle. We found that MG patients produce fundamentally the same pattern of specificities as that produced by animals immunized with receptor purified from fish electric organs or mammalian muscle. Most of the antibodies are directed at the "main immunogenic region' which is located on the extracellular surface of the alpha subunit and is distinct from the acetylcholine binding site.

Mapping of surface structures of electrophorus acetylcholine receptor using monoclonal antibodies.

Forty monoclonal antibodies to acetylcholine receptor from the electric organs of Electrophorus electricus have been characterized by immunoglobulin isotype, affinity for receptor, and specificity for species, subunit, and determinants within subunits. Using these antibodies, nine immunogenic regions on the receptor molecule were distinguished. Most of these are species specific, and are located on various subunits of the acetylcholine receptor.

Monoclonal antibodies as probes of acetylcholine receptor structure. 2. Binding to native receptor.

Binding of monoclonal antibodies top Torpedo californica acetylcholine receptor monomers solubilized in Triton X-100 was studied by centrifugation on sucrose gradients. Antibodies to alpha subunits were of two types. One type formed complexes of one antibody and one receptor monomer, independent of antibody/receptor ratio.

Monoclonal antibodies as probes of acetylcholine receptor structure. 1. Peptide mapping.

The isolated subunits of the acetylocholine receptor from Torpedo californica were digested with proteolytic enzymes, and the resulting polypeptide fragments were analyzed by gel electrophoresis. We have identified those fragments which contain carbohydrate and those from the alpha subunit which are labelled with the acetylcholine binding site specific reagent [4-(N-maleimido)benzyl]tri[3H]methylammonium iodide. We have tested several monoclonal antibodies raised to the acetylcholine receptor from torpedo, some of which react with the denatured subunits [Tzartos, S.

Investigation of sperm receptors in the hamster zona pellucida by using univalent (Fab) antibodies to hamster ovary.

An antiserum raised in rabbits against hamster ovaries was digested with papain to obtain the univalent (Fab) fragments. The anti-zona pellucida activity of this preparation was monitored by a direct fluorescence test and by an indirect precipitation test. In contrast to the effects produced by immune gamma-globins on the zona pellucida, the immune Fab antibodies failed to form a precipitate layer on the zona pellucida and did not inhibit fertilization in vitro.