Monoclonal antibodies to Torpedo acetylcholine receptor. Characterisation of antigenic determinants within the cholinergic binding site

Thirteen monoclonal antibodies (mAb) to the acetylcholine receptor (AChR) from Torpedo marmorata showed high avidity for the receptor but none exhibited binding to muscle AChR solubilised from seven other animal species. Five mAb and Fab monomer fragments prepared from two of them, inhibited alpha-bungarotoxin (alpha BuTx) binding to receptor by a maximum of 50%. In the presence of excess mAb the 125I-alpha BuTx bound could be precipitated by anti-IgG indicating that the mAb bound to only one of the two alpha BuTx binding sites on each AChR monomer. This site appeared to have a lower affinity for d-tubocurarine and decamethonium than the non-mAb site. Binding of five anti-site mAb was mutually competitive and four of them (AS2-AS5) were inhibited by other cholinergic ligands and influenced by four non-toxin binding site antibodies. One (AS1) bound within the toxin binding site yet outside the main neurotransmitter binding region. It is concluded that these five mAb distinguish between the two alpha BuTx binding sites on the Torpedo AChR, and bind only to the site which displays lower affinity for d-tubocurarine and other competitive ligands.     

Characterization of rat prothymocyte with monoclonal antibodies recognizing rat lymphocyte membrane antigenic determinants

Utilizing the technique of fluorescence-activated cell sorting and monoclonal antibodies directed at rat membrane antigens, various subpopulations of Lewis bone marrow cells were isolated and subsequently transfused into sublethally irradiated, histocompatible NBr recipient rats by either intravenous or intrathymic inoculation. Recipient rats were sacrificed and cell suspensions from thymus and other lymphoid tissue were examined for the presence of the RT7.1 marker on Lewis thymus-derived lymphocytes by fluorescence-activated cell analysis. From these studies, the population of Lewis bone marrow cells that could reconstitute T cells in the NBr rats was found to be Ox-22 negative, Ox-7 positive, W3/13 positive, and Ox-18 positive. Further analysis characterized the prothymocyte as being Ox-7 upper 20% positive and W3/13 weakly positive. In addition this marrow cell population was able to protect lethally irradiated Lewis rats (9.5 Gy) in 30-day survival tests. These studies have indicated that the prothymocyte either has been derived from the Ox-22 negative, Ox-7 upper 20% positive, and W3/13 positive marrow cells or, like the hematopoietic stem cell, this cell has also been characterized by this phenotype.     

Interactions of acetylcholine receptor and acetylcholinesterase with lipid monolayers

The interaction of acetylcholine receptor and acetylcholinesterase with lipid monolayers was followed by measuring changes in surface pressure.When injected into the subphase of a lipid monolayer, the proteins caused increases in surface pressure from 5 to 10 dynes/cm, indicating a penetration of protein into the monolayer. At pH values below the isoelectric point of the proteins the incorporation was improved. The same was observed when Ca²⁺ (2 mM) was added.The presence of the enzyme in the mixed film could be demonstrated by using diiso[³H]propyl fluorophosphate-labelled acetylcholinesterase as well as by measuring enzyme activity. Acetylcholine receptor was shown to be present in the mixed film by using a complex made of the receptor and α-[³H]neurotoxin.     

Properties of monoclonal antibodies to nicotinic acetylcholine receptor from chick muscle

Four stable, hybrid-cell lines secreting monoclonal antibodies to distinct determinants on the nicotinic acetylcholine receptor from chick muscle have been established. These were characterised by the following criteria: immunoglobulin isotype, ability to produce experimental autoimmune myasthenia gravis in mice and reactivity towards homologous and heterologous acetylcholine receptor proteins. Two monoclonal antibodies were found to inhibit the reaction of alpha-bungarotoxin with homologous acetylcholine receptor; in addition one of these, on binding to receptor-toxin, induced a rapid dissociation of the complex (t1/2 = 0.5 h at 23 degrees C). Three of the antibody preparations recognised epitopes on this receptor from muscle of other species and two of these caused experimental autoimmune myasthenia gravis in BALB/c mice following passive transfer. The latter two recognised to significant extents the alpha-bungarotoxin binding component purified from chick optic lobe and brain cortex. Sedimentation analysis demonstrated that two of the monoclonal antibodies form a distinct size (s20, w = 12S) of complex with the receptor of chick muscle which most probably corresponds to a 1:1 attachment of antibody and receptor; this may involve cross-linking of two determinants within the same oligomer. A similar observation was made with the alpha-bungarotoxin binding component from optic lobe using one of the cross-reacting antibodies. Another monoclonal antibody was found to be capable of forming much heavier complexes with the receptor from chick muscle, these are thought to involve inter-molecular cross-linking of oligomers. The observed properties of these antibodies are discussed in relation to their myasthenogenicity and with reference to the extent of structural similarities between the peripheral nicotinic acetylcholine receptor and the alpha-bungarotoxin binding protein from brain.     

Studies of nicotinic acetylcholine receptor protein from rat brain

Specific binding of ¹²⁵I-labeled α-bungarotoxin to a 34 800 × g pellet of a whole rat brain homogenate has been obtained at levels 2 pmol toxin per g of whole brain with a K_d of 8·10^?9 M. Binding is reduced 90% by 10^?5 M (+)- tubocurarine chloride and 10^?4 M nicotine, whereas concentrations of 10^?4 M choline chloride, atropine sulfate and eserine sulfate have essentially no effect on toxin binding. These results compare closely with those obtained from binding studies with ¹²⁵I-labeled α-bungarotoxin and soluble acetylcholine receptor protein preparations form Torpedo nobiliana; suggesting that this mammalian receptor protein is nicotinic in character.Extraction of the 34 800 × g pellet with 1% Emulphogene yields a soluble fraction with specifically binds ¹²⁵I-labeled α-bungarotoxin with a K_d of 5·10^?9 M. Nicotine and α-bungarotoxin at concentrations of 10^?5 M abolish toxin- receptor complex formation and carbachol and (+)-tubocurarine chloride reduce complex formation 35–40% at similar concentrations. Eserine sulfate, atropine sulfate, decamethonium, and pilocarpine had no effect on complex formation at concentrations of 10^?5 M.     

Location of antigenic determinants on primary sequences of subunits of nicotinic acetylcholine receptor by peptide mapping

The binding domains of 28 monoclonal antibodies (mAbs) against the alpha, beta, and delta subunits of the Torpedo acetylcholine receptor were mapped on the primary sequences of these subunits. Small peptide fragments (2000-20,000 daltons) of the purified subunits were obtained by digestion with staphylococcal V8 protease and papain, separated on a discontinuous polyacrylamide gel electrophoretic system, and electroblotted onto diaminophenyl thioether paper. The blots were probed with the various monoclonal antibodies and also with antibodies against carboxy-terminal decapeptides of the alpha, beta, and delta subunits to identify the carboxy-terminal fragments. From inspection of the binding patterns of the various antibodies to the subunits fragments and the molecular weights of these fragments, and by using the carboxy termini of the subunits as reference points, it was possible to deduce the regions on the primary sequence of each subunit in which the antibodies bound and in some cases to order the binding sites within these sequences. mAb 148, which inhibits receptor function by cross-linking receptor molecules on the cytoplasmic side, was mapped to the sequence beta 368-406. The main immunogenic region of the native receptor, which is of pathological importance in the autoimmune disease myasthenia gravis, was mapped by using mAb 210 to within 80 amino acid residues (alpha 46-127). The overall antigenic structure of alpha subunits was examined. Synthetic peptides have been used to locate determinants responsible for 83% of the antibodies in antisera to denatured alpha subunits and 46% of the antibodies to denatured alpha subunits in antisera to intact receptor. Theoretical models of the transmembrane orientation of the subunit polypeptide chains were tested by determining whether mapped monoclonal antibodies bound to the extracellular or intracellular surface of receptor-rich membranes. Our results confirm previous reports that the carboxy termini of the subunits are exposed on the intracellular surface, as is part of the region between a putative channel-forming domain (M5) and a putative membrane-spanning region (M3). However, contrary to current theoretical models, the region between M5 and the putative membrane-spanning sequence M4 also appears to be on the intracellular surface, implying that M4 and M5 are not membrane-spanning domains.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interactions of brain muscarinic acetylcholine receptors with plant lectins

We previously reported that muscarinic acetylcholine receptors (mAChRs) from porcine brains are glycoproteins. When porcine brain membranes were solubilized with digitonin or 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), approximately 20% of the receptors were solubilized, most (90% or more) of which bound to Sepharose 4B conjugated with wheat germ agglutinin (WGA). In contrast, when membranes were solubilized with Lubrol PX, a much larger fraction (approximately 60%) of the receptors were solubilized. However, about a third of this solubilized receptor population remained unbound to WGA-Sepharose even in the presence of an excess amount of the lectin-Sepharose. These results suggested a structural heterogeneity of the mAChR in terms of its carbohydrate moiety. The effects of lectins on the ligand binding properties of mAChRs were also studied. WGA or concanavalin A (ConA) was found to cause a 2- to 3-fold increase in the affinity of membrane-bound receptors to an antagonist [3H]quinuclidinyl benzylate [( 3H]QNB) without affecting the maximum number of sites, whereas the lectins had no significant effects on the binding of the agonist [3H]cis-methyldioxolane. When the membranes were dissolved with detergents, lectin did not increase the [3H]QNB affinity: These lectins caused an approximately 2 fold decrease in the affinity of digitonin-solubilized receptors for [3H]QNB. Thus the lectins exert differential effects on agonist and antagonist binding to the brain membrane mAChRs, most likely by modulating some intermolecular interactions.     

Monoclonal antibodies directed against different antigenic determinants of rotavirus

We have developed a series of monoclonal antibodies against the calf strain RIT 4237 (subgroup 1) and the human strain 82-561 (subgroup 3) of rotavirus, both grown in tissue culture, and also against the human rotavirus 81-2162 (subgroup 2), extracted from a fecal specimen. A variety of different specificities was observed among these antibodies, namely, antibodies against group and subgroup determinants, as well as neutralizing antibodies. By using monoclonal antibodies against the subgroup antigen in an enzyme-linked immunoassay system, the constant predominance of subgroup 2 viruses in humans was confirmed in 74 stools collected from children in Brussels who suffered a diarrheal illness between July 1981 and June 1983. The availability of these antibodies also made it possible to improve the sensitivity and the specificity of the test system.     

Muscarinic acetylcholine receptor from rat brain. Partial purification and characterization.

A protein capable of binding atropine and (3H)propylbenzilylcholine mustard was solubilized and purified (200-fold) from rat brain. Pronase and trypsin, but not phospholipases, diminished the binding capacity of the solubilized receptor. The molecular weight of the salt-solubilized receptor as determined by gel filtration in the absence of detergents is 30,000. The purified protein showed specificity of binding toward muscarinic ligands. the high and low affinity dissociation constants of the receptor.atropine complex are 0.3 nM and 0.15 muM. Binding of atropine is pH-dependent with an optimum at 7.1. Ca2+ influences the binding of atropine and maximal binding occurs at 0.5 mM Ca2+. The subcellular distribution of the receptor was also examined.     

Highly purified snRNPs retain antigenic determinants towards anti Sm antibodies

snRNPs purified by centrifugation in density gradients (BRUNEL et al. Nucl. Acids. Res., 1981, 9, 815–830 and SRI-WIDADA et al. Biochim. Biophys. Res. Commun., 1982, $\text{104}$, 457–462) retain an important part (46%) of their antigenicity towards anti-Sm antibodies indicating that antigenic determinants are among the small 4–5 polypeptides between 9 and 14,000 daltons found to be tightly associated with snRNAs. Furthermore, digestion of a great part of the RNA moiety does not abolish this antigenicity.As to the determinant for RNP antigen, our results suggest that it could depend on the three other proteins which are lost during the purification.     

Influence of phospholipase C on muscarinic acetylcholine receptor binding in rat brain

Treatment of neural membranes from rat cerebral cortex with phospholipase C (phosphatidylcholine cholinephosphohydrolase) inhibited the binding of radiolabelled antagonists to muscarinic acetylcholine receptors. This inhibition was incomplete, was not competitive, and did not appear to be related to the production of inhibitory products. The affinity of carbamylcholine for cortex muscarinic receptors was increased by phospholipase C action. The distribution of receptors between states of high and low affinity was not affected by phospholipase C; rather, the affinity for carbamylcholine of the lowest affinity receptors was selectively increased. This suggests that membrane lipids influence the interaction of the receptor binding subunit with other structures in the synaptic membrane.     

Evolution of HLA antigenic determinants: Species cross-reactions of monoclonal antibodies

The reactions of mouse monoclonal HLA-A-, -B-, -C-specific antibodies on cells from 50 species were analyzed by binding assay. Antibodies which are monomorphic in humans generally show monomorphic behavior in other species and many significant cross-reactions were seen. Antibodies which recognize highly specific polymorphic determinants gave few cross-reactions with other species. Antibodies which recognize broad polymorphic determinants in humans cross-reacted in a polymorphic manner with many other species. The patterns of cross-reaction for both monomorphic and broadly polymorphic determinants correlated roughly with the current picture of phylogenetic relationships. These results show there are two fundamentally different classes of polymorphic determinants, one which appears relatively conserved in evolution and one which is the product of recent change. They probably correlate with the public and private antigens of mouse H-2 antigens. A simple genetic model ofHLA-A, -B, -C genes, whereby all specificities at a locus are true alleles, is sufficient to explain these patterns of cross-reaction. It seems likely that cross-reactions previously seen with alloantisera were due to broadly polymorphic antibodies rather than to allele-specific antibodies. If this were the case, then no experimental basis for Bodmer's model of MHC polymorphism being due to control of expression of a tandem array of genes exists.Abbreviations used in this paper MHC major histocompatibility complex - PBS phosphate-buffered saline - BSA bovine serum albumin - RAM rabbit anti-mouse IgG F(ab)₂ fragments     

Solubilization of muscarinic acetylcholine receptor by zwitterionic detergent from rat brain cortex

The muscarinic acetylcholine receptor was solubilized from rat brain cortex by zwitterionic detergent 3-[(3-chloramidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS). About 15% of the binding activity was solubilized and 40% of the activity was destroyed by the detergent. Binding of the muscarinic antagonist [³H]-N-methyl-4-piperidyl benzilate (4NMPB) was saturable. Scatchard analysis revealed a single population of binding sites with K_D value of 0.7 nM and a B_max value of 340 fmoles/mg protein. The homogenate and the CHAPS treated pellet and soluble receptors showed similar affinity for the agonists oxotremorine and carbamylcholine and for the antagonists QNB and atropine. The dissociation of 4NMPB from the soluble receptors appears slightly slower than from the membrane bound receptors.     

The antigenic structure of the acetylcholine receptor from Torpedo californica

The immunological structure of the acetylcholine receptor (AChR) from the electric organ of Torpedo californica was studied using a large number of monoclonal antibodies which were initially selected for their abilities to bind to intact AChRs. The monoclonal antibodies were tested for their ability to bind to denatured AChR subunits labeled with 125I. Antibodies derived from rats immunized with individual denatured subunits or a mixture of subunits of Torpedo AChR reacted well in the assay. A much smaller proportion of antibodies derived from rats immunized with native Torpedo AChR or native AChR from Electrophorus electricus electric organ, bovine muscle, or human muscle reacted with denatured subunits of Torpedo AChR. Many monoclonal antibodies reacted with more than one subunit, but they always reacted best with the subunit used for immunization. Those monoclonal antibodies that bound to intact subunits were mapped more precisely by their ability to bind characteristic fragments of each subunit generated by proteolysis with Staphylococcal V8 protease. These fragments were analyzed by SDS polyacrylamide gel electrophoresis, and monoclonal antibodies that precipitated the same fragment pattern were placed in groups. By this method, we define a minimum of 28 determinants on Torpedo AChR.     

The identification of antigenic determinants on Mycobacterium bovis using monoclonal antibodies

Murine hybridomas secreting monoclonal antibodies to Mycobacterium bovis were produced and three soluble antigens were identified using radioimmunoassays and immunoblotting from polyacrylamide electrophoresis gels. Antibody MB3 (IgM, k chain) reacted with 20-100 kDal antigens produced by all mycobacterial strains examined while antibody MB5 (IgG2a, k chain) identified a 29.8 kDal antigen detected in field isolates of M. bovis and M. bovis strains Vallée and AN5. There was insignificant binding to M. bovis BCG, M. tuberculosis, M. microti, M. africanum, M. avium or M. paratuberculosis. Monoclonal antibody MB17 (IgA, k chain) reacted with a 17.4 kDal antigen present in M. bovis, M. tuberculosis and M. microti. Absorption of monoclonal antibodies with antigens from different species of Mycobacterium confirmed the specificities of MB3 and MB5 but the binding of MB17 was inhibited to some extent by all the extracts examined. The antigen identified by MB3 was present in purified protein derivative (PPD) from M. bovis, M. paratuberculosis and M. avium but antigens identified by MB5 and MB17 were not detected in these reagents.     

Monoclonal antibodies against the human acetylcholine receptor

Monoclonal cell lines synthesizing antibodies against partially purified acetylcholine receptor from human muscle (H.AChR) were produced. Eleven clones secreted antibodies against H.AChR. Four were obtained in ascitic form. Two of them have been exhaustively studied. Specificity and affinity for H.AChR were demonstrated. Cross-reactivity with mouse AChR was shown but not with torpedo or porcine AChR at the same concentration. Purified IgG injected intravenously provoked an obvious muscular weakness. Inhibition experiments on myasthenia gravis sera binding have demonstrated that monoclonal antibody specificity is directed against an antigenic determinant shared by human and mouse AChR.