Use of monoclonal antibodies to study acetylcholine receptors from electric organs, muscle, and brain and the autoimmune response to receptor in myasthenia gravis.
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Biomedical subjects
J Lindstrom
Publications and source records attributed to J Lindstrom.
The regulation of acetylcholine receptor expression in mammalian muscle.
The synthesis of functional AChRs can be described as a pathway leading from the translation of subunit mRNAs to the plasma membrane forms of extrajunctional and junctional receptors (Fig. 9). We have not included in this scheme pretranslational steps for the synthesis and processing of RNA coding for receptor subunits because very little is known about such processes. Several aspects of Figure 9 are worthy of note: It is now well established that polypeptide synthesis is initiated on free cytoplasmic polysomes and that once sufficient nascent subunits bearing signal peptides at the amino terminus is formed, polysomes assemble with the membranes of the rough endoplasmic reticulum via a mechanism that employs the signal recognition particle (Anderson et al. 1982). Nascent subunits undergo cotranslational insertion through the rough endoplasmic reticulum membrane, signal peptide removal, and core glycosylation (Anderson and Blobel 1981; Merlie et al. 1981; Anderson et al. 1982; Sebbane et al. 1983). Anderson and Blobel (this volume) have demonstrated that subunits synthesized in vitro and inserted into membrane vesicles do not undergo heterologous subunit-subunit associations. We have shown that alpha- and beta-subunits newly synthesized in vivo are not associated with each other. Our data indicate that the alpha-subunit is initially present in vivo in a conformation that is radically different from its native conformation in the mature receptor complex. We assume that beta-, gamma-, and delta-subunits also are synthesized as conformationally "immature" forms, but verification of this point must await the availability of new monoclonal antibody specificities. Our data indicate that only a fraction of the newly synthesized alpha-subunit undergoes conformational maturation to the 5S species which binds both alpha-bungarotoxin and anti-main immunogenic region monoclonal antibodies. alpha-Subunits synthesized during a 5-minute pulse labeling require 30 minutes for completion of this process. alpha-Subunits that do not undergo conformational maturation are degraded rapidly (t1/2 = 0.5 hr) ( Merlie et al. 1982). Assembly of alpha- and beta-subunits synthesized during a 5-minute pulse labeling lags for approximately 30 minutes and is not complete until 90 minutes. Finally, assembled receptors are transported to the surface and appear in the plasma membrane. These processes occur during expression of AChRs in differentiated myoblasts. We do not know how undifferentiated myogenic cells, in vivo or in tissue culture, differ with regard to any of these steps.(ABSTRACT TRUNCATED AT 400 WORDS)
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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.J., P. Walter, and G. Blobel (1982) J. Cell Biol. 93: 501-506). Here we demonstrate that this domain can be further dissected into a 26-kd fragment, together with low molecular weight material, when the membranes are rendered permeable to trypsin by low concentrations of deoxycholate (Kreibich, G., P. Debey, and D. D. Sabatini (1973) J. Cell Biol. 58: 436-462). This 26-kd fragment contains all of the core oligosaccharides present on the intact subunit and therefore constitutes at least part, if not all, of the extracellular domain. The remaining low molecular weight material may derive from the membrane-embedded domain; our data imply that as much as 18 kd may be internal to the lipid bilayer. On the other hand, part of the cytoplasmic pole of AChR-delta can be recovered as a discrete, 12-kd fragment upon mild trypsinization of intact vesicles. We have used this 12-kd fragment to identify anti-AChR-delta monoclonal antibodies (mAbs) that react with the cytoplasmic domain of this subunit. Partial proteolytic fragmentation of the AChR in vitro translation products, in topologically well defined rough microsomes, may be used as a general assay to characterize the domain specificity of anti-AChR mAbs. For example, in the case of AChR-beta, we were able to identify two mAbs that recognize extracellular and cytoplasmic fragments, respectively.
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Subunit composition of bovine muscle acetylcholine receptor.
Acetylcholine receptors from fetal calf muscle were purified to homogeneity (specific activity up to 7500 nmol/g of protein), in reasonable yields (20-50%), and near-milligram quantity. Purification was by affinity chromatography on Naja naja siamensis toxin coupled to agarose by using methods similar to those for receptors from fish electric organs, but with modifications to account for the low concentration of receptor in muscle and the high probability of proteolysis. Immunochemical methods are described for approximating the extent of proteolysis in receptor preparations. Bovine acetylcholine receptor is composed of four glycoprotein subunits designated alpha (Mr congruent to 41 000), beta (Mr congruent to 50 000), gamma (Mr congruent to 53 000), and delta (Mr congruent to 56 000) which correspond immunochemically to the four glycoprotein subunits of fish electric organ acetylcholine receptors of the same designations. Electron micrographs of purified bovine receptor show that it has the same size and shape as receptors from fish electric organs. Immunization of rats with receptor from bovine and human muscle is very effective at inducing experimental autoimmune myasthenia gravis. Acetylcholine receptors purified from rat muscle are composed of subunits which correspond immunochemically to the alpha, beta, gamma, and delta subunits of receptor from Torpedo californica. The evidence presented strongly suggests that acetylcholine receptors from fish electric organ tissue and mammalian muscle share a fundamentally similar shape, antigenic structure, and alpha 2 beta gamma delta subunit structure.
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Incorporation of acetylcholine receptors into liposomes. Vesicle structure and acetylcholine receptor function.
Functionally intact acetylcholine receptors can be solubilized from electric organ membranes of Torpedo californica and incorporated into liposomes by the cholate dialysis technique. Freezing and thawing of the reconstituted preparation appears to seal a population of initially leaky vesicles and leads to vesicle fusion. Inclusion of supplementary cholesterol at an optimal concentration of 20% (w/w) greatly enhances vesicle fusion during the freeze-thaw cycle. Size analysis by electron microscopy of negatively stained preparations indicates that fusion is accompanied by shifts in size and volume distributions of the vesicle population. Liposomes formed in the absence of acetylcholine receptors are distributed over a substantially smaller size range than liposomes containing receptors. Acetylcholine receptors appear in those liposomes as dimers of 80 A doughnut-shaped particles. Freeze-fracture replicas of reconstituted preparations reveal the presence of large vesicles containing particles which correspond in size to acetylcholine receptors and smaller liposomes devoid of particles. The distribution of particles in the reconstituted membranes is sparse compared to their dense packing in native electric organ membranes. The activation and desensitization of reconstituted acetylcholine receptors mediated by acetylcholine or carbamylcholine is dose dependent. The reconstituted receptors distinguish between these agonists in terms of binding affinity in a way similar to receptors in the native membrane. Correlation of the fractional occupancy of ligand binding sites by cobratoxin with inhibition of receptor function is used to demonstrate that in the reconstituted system the doubly liganded acetylcholine receptor prevails in controlling channel gating. The potential experimental advantages as well as limitations of this reconstituted system are discussed.
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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. We have investigated the role of glycosylation in the biogenesis of the acetylcholine receptor. Receptor biogenesis in normal cells involves the assembly of newly synthesized alpha subunits into a form active for binding alpha-bungarotoxin. This process is only 30% efficient and is complete by 30 min postsynthesis. When glycosylation is inhibited by tunicamycin, alpha subunit synthesis is inhibited only slightly but assembly into an alpha-bungarotoxin binding species is reduced dramatically.
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Acetylcholine receptor antibody titer and HLA-B8 antigen in myasthenia gravis.
In 82 white patients with myasthenia gravis, a high serum human acetylcholine receptor (AChR) antibody titer was related to the presence of the HLA-B8 antigen and increasing severity of the disease and not to age at onset, sex, presence of thymoma, or mode of treatment. Among patients without thymomas a high antibody titer was also associated with HLA-B8, particularly in those patients whose age at onset was less than 35 years. Thymectomy was associated with a lower median antibody titer when compared in two groups of HLA-B8-positive patients without thymoma who were similar for all other factors. Patients with thymomas who had received corticosteroids had a lower median titer than those who had not received steroids. This study supports the possibility that immune-response genes near the HLA-B8 segment of the major histocompatibility complex participate in the regulation of the humoral response to autoantigens, such as AChR protein.
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Leukoplasmapheresis for myasthenia gravis. Acetylcholine receptor antibody production.
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Adult↗
Ultrastructural aspects of acetylcholine receptor turnover at the normal end-plate and in autoimmune myasthenia gravis.
Acetylcholine receptor (AChR) deficiency at the myasthenic end-plate has been attributed to complement-mediated lysis of the junctional folds and to increased fractional degradation rate of AChR cross-linked by myasthenic immunoglobulin. This paper addresses the manner in which AChR is internalized and degraded at the normal end-plate and provides morphologic evidence for accelerated AChR degradation at the end-plate of rats with experimental autoimmune myasthenia gravis (EAMG). We sequentially traced the fate of end-plate AChR labeled in vivo with intramuscularly-injected peroxidase-alpha-bungarotoxin (PBGT) in control rats and rats with chronic EAMG. At both control and EAMG end-plates, AChR is internalized by endocytosis. The endocytosed vesicles containing AChR are transferred into the lysosomal compartment which extends from the junctional folds into the junctional sarcoplasm. Regardless of whether the initial intensity of the reaction for AChR at the EAMG end-plate appeared normal or reduced. AChR disappeared more rapidly from the EAMG than from the control end-plates. Despite the accelerated fractional turnover rate of end-plate AChR in EAMG, the postsynaptic membrane surface which could be labeled with PBGT for AChR remained unchanged over a 120-hour period. These data suggest that end-plate AChR is at a steady state in chronic EAMG.
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Estimation of degradation rate of acetylcholine receptor by external gamma counting in vivo.
We describe a novel method for estimating the rate of destruction of the acetylcholine receptor (AChR) associated with the motor end-plate in the living animal. After the intramuscular injection of a nontoxic dose of alpha-bungarotoxin that had been monoiodinated with 125I (125I-BGT), the loss of radioactivity from the injected site is monitored by an externally positioned gamma counter. Two days after labeling, the decline of radioactivity in the injected muscle parallels the decline of end-plate specific radioactivity and can be used for the estimation of the degradation rate and half-life of end-plate AChR. Using this method, we measured AChR half-life in normal and in myasthenic rats and found a 2.5-fold decrease of AChR half-life in the myasthenic animals. This in vivo method has potential clinical applications.
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Stabilization of acetylcholine receptor channels by lipids in cholate solution and during reconstitution in vesicles.
Acetylcholine receptors were solubilized from electric organ membranes of Torpedo californica in mixed micelles of sodium cholate and soybean lipids. Sodium cholate, when supplemented with relatively low amounts of soybean lipids (cholate:lipid, 20:1, molar ratio), was effective in solubilizing receptors without denaturing their agonist-regulated cation channels. Another dialyzable detergent, octylglucoside, denatured the ion channel even in the presence of excess lipids. Reassembly of receptors and lipids into vesicles was achieved by cholate dialysis. About 70% of the receptors were oriented with their toxin binding sites on the external surface of the vesicles. Evidence suggests that all of the receptors in a single vesicle were oriented either right side out or inside out. During the reassembly process about 10-fold greater lipid concentrations were required for the preservation of channel function. At lipid/protein ratios greater than 16:1 (w/w), receptors reassembled into vesicles at a constant protein/lipid ratio. These vesicles contained approximately 7% receptors by weight, 5-fold less than the native membrane. The remainder of the lipid assembled into small vesicles which did not contain receptors. At lipid/protein ratios less than 16:1 (w/w), receptors reassociated with lipids in higher weight ratios. Irreversible inactivation of a fraction of the acetylcholine receptor channels occurred in proportion to the greater packing density. This channel denaturation was accompanied by a lowered susceptibility of the disulfide bond between the delta subunits of the acetylcholine receptor dimer to reducing agents. Toxin binding and the orientation of the receptors in the reconstituted vesicles was not affected by reduced lipid/protein ratios. The unexpected constant acetylcholine receptor/lipid ratio in the reconstituted vesicles and the unexpected uniform orientation of the acetylcholine receptors within a vesicle are discussed in terms of the interactions occurring during the initial nucleation events of the reassembly process.
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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.J., & Lindstrom, J.M. (1980) Proc. Natl. Acad. Sci. U.S.A.77, 755; Tzartos, S.J., & Lindstrom, J.M. (1981) in Monoclonal antibodies in Endocrine Research (Fellows, R., & Eisenbarth, G., Eds.) Raven Press (in press)]. The binding specificities of these antibodies to radioiodinated proteolytically generated fragments of the alpha subunit were determined by immunoprecipitation followed by gel electrophoresis. The antibodies tested fell into at least three main groups on the basis of their binding specificities. These antibodies were also tested for their capacity to bind to acetylcholine receptor solubilized in Triton X-100, sodium cholate, or sodium cholate supplemented with exogenous lipids. A monoclonal antibody raised to the denatured delta subunit, was tested for its ability to select radioiodinated proteolytic fragments of these subunits. These molecules provide probes for many sites on the acetylcholine receptor with affinities and specificities comparable to alpha-neurotoxins.
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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. We conclude that the binding sites for these antibodies are oriented on the two alpha subunits per monomer in such a way that each could be bound by one of the two binding sites of a single immunoglobulin molecule. Most antibodies were of this type. The other type of monoclonal antibody formed complexes of several sizes, including antibody cross-linked receptors, depending on the ratio of antibody to receptor. We conclude that the binding sites for these antibodies are oriented in such a way that the two alpha subunits per monomer could not be cross-linked by a single antibody molecule. A monoclonal antibody of this type raised against Electrophorus electricus receptors was used to show that this receptor also has two alpha subunits per monomer. This antibody cross-reacted with receptor from fetal calf muscle and was able to induce modulation of receptor in muscle cells in culture. This suggests that muscle receptor also has two alpha subunits and that the antibody can cross-link receptor in the plane of the membrane, as it does in solution, and thereby form complexes which enhance endocytosis and increase the rate of receptor destruction. The rate of antigenic modulation decreases at high antibody/receptor ratios, as expected if un-cross-linked complexes of two antibodies and one receptor were not destroyed at a faster rate. Antibodies which cross-link alpha subunits within a receptor monomer are frequent but would not be expected to be able to induce antigenic modulation. This provides one mechanism by which antisera of equivalent antireceptor titer might differ in their ability to induce antigenic modulation. An antibody which binds to denatured delta and gamma subunits forms complexes of only one antibody and one receptor monomer, independent of antibody ratio, as do antibodies thought to cross-link the two alpha subunits in a monomer. It apparently cross-links delta and gamma subunits within the monomer. Some of the monoclonal antibodies to alpha subunits can bind simultaneously to receptor, while the binding of others is mutually exclusive.
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Production and assay of antibodies to acetylcholine receptors.
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Structure and function of the acetylcholine receptor molecule studied using monoclonal antibodies.
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Antibodies to acetylcholine receptors as probes of receptor structure and pathological agents in myasthenia gravis.
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Animals↗
Antibodies to polyadenylic acid in patients with myasthenia gravis.
Sera from 100 patients with myasthenia gravis and 45 patients with non-myasthenia gravis neuromuscular diseases were studied for antibodies to poly rA, poly rA-rU, native and denatured DNA. All patients with myasthenia gravis had significant anti-acetylcholine receptor antibodies with a mean titre of 1.2 X 10(-7)M. Forty-eight per cent of the myasthenia gravis patients had anti-poly rA antibody levels which were greater than 3 standard deviations from the mean of 65 control patients by Millipore filter radioimmunoassay. The antibody was specific for poly rA and present in a much higher frequency than antibodies to the other nucleic acids tested. Sucrose-gradient ultracentrifugation demonstrated that the antibody was limited to the IgM class alone. Mechanisms relating these findings to a more generalized immunological dysfunction are discussed.
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Proteolytic nicking of the acetylcholine receptor.
Low concentrations of papin rapidly cleave solubilized or membrane-bound acetylcholine receptor (AcChR) from Torpedo californica into a wide range of small fragments. The alpha subunits of the receptor are most resistant to cleavage. After solubilization in sodium dodecyl sulfate solutions the fragments are dissociated, and on electrophoresis the apparent subunit composition is reduced from four types (alpha, beta, gamma, and delta) to only alpha and finally, with large amounts of papain, to fragments even smaller than alpha. Prior to dissociation in sodium dodecyl sulfate, the proteolytic fragments remain physically and functionally associated. Thus, receptor which has been degraded so as to apparently contain only alpha subunits, or even no obvious subunits, still retains antigenic determinants corresponding to each subunit, still retains its characteristic size and doughnut shape when examined electron microscopically, and still sediments as dimers on sucrose gradients. Moreover, proteolytically nicked receptor remains fully functional in carbamylcholine-induced 22Na+ flux. These results demonstrate that inadequate inhibition of proteases during purification of receptor could account for reports from some laboratories that they have purified receptors containing only alpha subunits or fragments of alpha subunits. Also, our results demonstrate the strong noncovalent association between AcChR subunits which has thus far precluded their separation except under denaturing conditions in sodium dodecyl sulfate.
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