Localization of the functional sites on the alpha chain of acetylcholine receptor

A comprehensive synthetic approach, previously developed in this laboratory, has been applied to systematically screen the entire extracellular part (residues 1-210) of the alpha chain of the Torpedo californica acetylcholine receptor (AChR) for the profiles of the continuous regions that are recognized by antibodies against free, or membrane-sequestered, AChR; the regions recognized by AChR-primed T cells; the regions that bind alpha-bungarotoxin and cobratoxin; and an acetylcholine-binding region. Eight continuous antigenic sites were localized in this part of the alpha chain by all of the antisera tested. The sites were independent of the host species from which the antisera were obtained and were also similar to antisera against the isolated pentameric AChR or against the membrane-sequestered AChR. Six regions were found to stimulate AChR-primed T cells (T sites). Three of the T sites coincided with regions recognized by antibodies. At least two T sites had no detectable antibody responses directed to them. Five toxin-binding regions were localized, and may constitute distinct sites or, alternatively, different faces in one (or more) sites. Some of these regions coincided with regions recognized by anti-AChR antibodies. One of the toxin-binding regions bound acetylcholine, and immunization with this peptide induced experimental autoimmune myasthenia gravis.     

α-Neurotoxin binding to acetylcholine receptor: Localization of the full profile of the cobratoxin-binding regions on the α-chain ofTorpedo californica acetylcholine receptor by a comprehensive synthetic strategy

Eighteen consecutive uniform overlapping synthetic peptides that spanned the entire extracellular part (residues 1–210) of the α-chain ofTorpedo californica acetylcholine receptor were screened for binding activity of¹²⁵I-labeled cobratoxin. Five toxin-binding regions were localized within residues 1–10, 32–41, 100–115, 122–150, and 182–198. The five toxin-binding regions may be distinct sites or, alternatively, different faces in one or more sites.     

Segment alpha 182-198 of Torpedo californica acetylcholine receptor contains second toxin-binding region and binds anti-receptor antibodies

The area around Cys-192 and Cys-193 is thought to be a functionally important part of the alpha-subunit of the acetylcholine receptor. We have synthesized peptide alpha 182-198 of the alpha-chain of the Torpedo californica acetylcholine receptor and investigated the binding to the peptide of alpha-bungarotoxin, cobratoxin and antibodies raised against acetylcholine receptor. The results showed that the synthetic peptide alpha 182-198 contains a second toxin-binding region and also binds a considerable fraction of anti-receptor antibodies. We also report here the toxin-binding activity of synthetic peptide alpha 125-148 of the human acetylcholine receptor which has been previously localized as a toxin-binding region in the alpha-chain of the Torpedo receptor.     

The regions of α-neurotoxin binding on the extracellular part of the α-subunit of human acetylcholine receptor

A set of 18 synthetic uniform overlapping peptides spanning the entire extracellular part (residues 1–210) of the α-subunit of human acetylcholine receptor were studied for their binding activity of¹²⁵I-labeled α-bungarotoxin and cobratoxin. A major toxin-binding region was found to reside within peptide α122–138. In addition, low-binding activities were obtained with peptides α34–49 and α194–210. It is concluded that the region within residues α122–138 constitutes a universal major toxin-binding region for acetylcholine receptor of various species.     

Localization of the metal-induced conformational transition of bovine prothrombin

The calcium-stabilized antigenic determinants on bovine prothrombin were localized to the NH2-terminal 1-42 residues using conformation-specific antibodies. Polyclonal antibodies to the bovine prothrombin-Ca(II) complex were raised in rabbits, and purified antibody subpopulations were isolated by sequential immunoabsorption and affinity chromatography. Anti-prothrombin-Ca(II) antibodies, characterized by their absolute specificity for the prothrombin-metal complex (Tai, M. M., Furie, B. C., and Furie, B. (1980) J. Biol. Chem. 255, 2790-2795), bound to prothrombin, fragment 1, reduced and carboxymethylated fragment 1, and CNBr fragment (1-72) in solution. However, these antibodies do not bind significantly to the gamma-carboxyglutamic acid-rich fragment (1-39), CNBr fragment (73-156), or prethrombin 1. To obviate the complex analysis of possible reasons for the lack of antibody binding to small peptides in solution, conformation-specific antibodies directed against defined regions of the whole prothrombin molecule were isolated. The influence of calcium ions on the binding of these site-specific antibody subpopulations to 125I-labeled prothrombin fragment 1 was evaluated. Anti-(1-39)N, anti-(1-42)N, anti-(1-72)N, and anti-(reduced and carboxymethylated fragment 1)N showed enhanced binding to prothrombin fragment 1 in the presence of Ca(II), indicating the presence of calcium-stabilized antigenic determinants within each of these regions on fragment 1. In contrast, calcium ions had no effect on the interaction of anti-des-(1-42)prothrombin, anti-prethrombin 1, anti-(43-72)N, and anti-(73-156)N antibodies with prothrombin fragment 1. These results indicate that the metal-induced conformational transition, monitored immunochemically, is localized to the NH2-terminal, gamma-carboxyglutamic acid-rich region of prothrombin between residues 1-42.     

Main Immunogenic Region of Torpedo Electroplax and Human Muscle Acetylcholine Receptor: Localization and Microheterogeneity Revealed by the Use of Synthetic Peptides

Most anti-nicotinic acetylcholine receptor (AChR) antibodies in myasthenia gravis are directed against an immunodominant epitope or epitopes [main immunogenic region (MIR)] on the AChR alpha-subunit. Thirty-two synthetic peptides, corresponding to the complete Torpedo alpha-subunit sequence and to a segment of human muscle alpha-subunit, were used to map the epitopes for 11 monoclonal antibodies (mAbs) directed against the Torpedo and/or the human MIR and for a panel of anti-AChR mAbs directed against epitopes on the alpha-subunit other than the MIR. A main constituent loop of the MIR was localized within residues alpha 67-76. Residues 70 and 75, which are different in the Torpedo and human alpha-subunits, seem to be crucial in determining the binding profile for several mAbs whose binding to the peptides correlated very well with their binding pattern to native Torpedo and human AChRs. This strongly supports the identification of the peptide loop alpha 67-76 as the actual location of the MIR on the intact AChR molecule. Residues 75 and 76 were necessary for binding of some mAbs and irrelevant for others, in agreement with earlier suggestions that the MIR comprises overlapping epitopes. Structural predictions for the sequence segment alpha 67-76 indicate that this segment has a relatively high segmental mobility and a very strong turning potential centered around residues 68-71. The most stable structure predicted for this segment, in both the Torpedo and human alpha-subunits, is a hairpin loop, whose apex is a type I beta-turn and whose arms are beta-strands. This loop is highly hydrophilic, and its apex is negatively charged. All these structural properties have been proposed as characteristic of antibody binding sites. We also localized the epitopes for mAbs against non-MIR regions. Among these, the epitope for a monoclonal antibody (mAb 13) that noncompetitively inhibits channel function was localized within residues alpha 331-351.     

Synthesis of an antigenic site of native acetylcholine receptor peptide 159-169 of Torpedo acetylcholine receptor alpha-chain.

A region of the alpha-subunit of the nicotinic acetylcholine receptor (AChR) of the Torpedo electric organ, containing residues 161-166, has been proposed to be a major antigenic site in the native AChR protein. We report the synthesis of a peptide corresponding to residues 159-169, which contains the proposed antigenic region. In quantitative radiometric titrations, radiolabelled anti-(native AChR) antibodies from three different species, rabbit, rat and dog, exhibited considerable binding (approx. 15% relative to native AChR) to Sepharose-immobilized peptide 159-169, but did not bind significantly to Sepharose-immobilized unrelated proteins or peptides. Specificity was further confirmed by the finding that no rabbit anti-AChR antibodies bound to the peptide after absorption with native AChR. These data indicate that the region 159-169 contains an antigenic site that is readily accessible in solubilized native Torpedo AChR.     

Production of antibodies to alpha(181-192) peptides of neuronal nicotinic acetylcholine receptor coupled to protein carriers in different orientations

The antibodies to nicotinic acetylcholine receptor alpha(181-192) synthetic peptides were elicited in rabbits and mice using the peptides conjugated to protein carriers in different orientations, either through C-terminal Cys (S-conjugates), or through amino groups (N-conjugates). S-conjugated peptides were less potent in eliciting peptide-specific antibodies compared to N-conjugates and this type of conjugation resulted in antibodies to the coupling reagent. However, the epitopes present in either S- or N-conjugated peptides appeared to be similar, indicating that amino acid residues, which form the epitope, were located in the middle part of the peptide and did not include both N- and C-terminal residues. Peptide conjugation to a protein carrier did not play a role in stabilizing the peptide conformation, but was necessary to concentrate the peptide epitopes on the carrier surface enabling bivalent antibody binding.     

Analysis of acetylcholine receptor phosphorylation sites using antibodies to synthetic peptides and monoclonal antibodies.

Three peptides corresponding to residues 354-367, 364-374, 373-387 of the acetylcholine receptor (AChR) delta subunit were synthesized. These peptides represent the proposed phosphorylation sites of the cAMP-dependent protein kinase, the tyrosine-specific protein kinase and the calcium/phospholipid-dependent protein kinase respectively. Using these peptides as substrates for phosphorylation by the catalytic subunit of cAMP-dependent protein kinase it was shown that only peptides 354-367 was phosphorylated whereas the other two were not. These results verify the location of the cAMP-dependent protein kinase phosphorylation site within the AChR delta subunit. Antibodies elicited against these peptides reacted with the delta subunit. The antipeptide antibodies and two monoclonal antibodies (7F2, 5.46) specific for the delta subunit were tested for their binding to non-phosphorylated receptor and to receptor phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. Antibodies to peptide 354-367 were found to react preferentially with non-phosphorylated receptor whereas the two other anti-peptide antibodies bound equally to phosphorylated and non-phosphorylated receptors. Monoclonal antibody 7F2 reacted preferentially with the phosphorylated form of the receptor whereas monoclonal antibody 5.46 did not distinguish between the two forms.     

Induction of antibodies to particular sites of the alpha7 subunit of the nicotinic acetylcholine receptor in mice of different lines

Five synthetic fragments of the N-terminal domain of the alpha7 subunit of the human nicotinic acetylcholine receptor (alpha7 nAChR) that correspond to theoretically calculated B epitopes and T helper epitopes of the protein and contain from 16 to 29 amino acid residues were tested for the ability to stimulate the formation of antibodies in mice of three lines having H-2d, H-2b, and H-2k haplotypes of the major histocompatibility complex. It was shown that, in the free (unconjugated) form, all the peptides stimulate the formation of antibodies at least in one mouse line. Most of the peptides induced the formation of antibodies in BALB/c mice (haplotype H-2d); therefore, more detailed studies were carried out on these animals. The free peptides and/or their conjugates with keyhole limpet hemocyanin were demonstrated to be capable of stimulating the formation in BALB/c mice of antibodies that bind to the recombinant extracellular N-terminal domain of (alpha7 nAChRalpha). The epitope mapping of antipeptide antibodies carried out using truncated fragments helped reveal antipeptide antibodies to four regions of the alpha7 subunit: 1-23, 98-106, 159-168, and 173-188 (or 179-188).     

Antigenic structure of human haemoglobin. Localization of the antigenic sites of the beta-chain in three host species by synthetic overlapping peptides representing the entire chain.

A comprehensive synthetic approach is applied here to localize the continuous antigenic sites of the beta-chain of haemoglobin. The approach was based on the synthesis and purification of the following consecutive 15-residue peptides (each overlapping by five residues at both ends with the peptides preceding it and following it in the sequence): 1-15, 11-25 etc. Quantitative radiometric titrations of protein and peptide adsorbents were performed with 125I-labelled anti-haemoglobin antibodies from three different host species. The specificity of antibody binding to peptide adsorbents was confirmed by inhibition studies and by the binding specificity of antibodies isolated from peptide adsorbents. These studies established the full profile of antigenic beta-chain regions, which was found to be independent of the host species. Five major antigenic sites were localized, and their three-dimensional and structural characteristics are discussed in relation to the immune recognition of haemoglobin and other proteins.     

The regions of alpha-neurotoxin binding on the extracellular part of the alpha-subunit of human acetylcholine receptor

A set of 18 synthetic uniform overlapping peptides spanning the entire extracellular part (residues 1–210) of the -subunit of human acetylcholine receptor were studied for their binding activity of¹²⁵I-labeled -bungarotoxin and cobratoxin. A major toxin-binding region was found to reside within peptide 122–138. In addition, low-binding activities were obtained with peptides 34–49 and 194–210. It is concluded that the region within residues 122–138 constitutes a universal major toxin-binding region for acetylcholine receptor of various species.     

Localization of a highly immunogenic region on the acetylcholine receptor alpha-subunit

Antibodies to synthetic peptides were employed in order to map domains on the alpha-subunit of the acetylcholine receptor to which several monoclonal antibodies are directed. Five peptides corresponding to residues 1-20, 126-143, 169-181, 330-340 and 351-368 of the receptor alpha-subunit were synthesized and antibodies against them were elicited. The anti-peptide antibodies were employed along with the monoclonal antibodies to identify fragments of S. aureus V8 protease digested- alpha-subunit in immunoblotting experiments. Our results demonstrate that a highly immunogenic region of the alpha-subunit is located on a carboxy-terminal 14 kDa portion of the alpha-subunit. This region also seems to undergo antigenic changes during muscle development. A monoclonal antibody directed against the cholinergic binding site of the acetylcholine receptor reacted with an 18 kDa segment of the alpha-subunit which bound alpha-bungarotoxin as well as antibodies directed against peptide 169-181.     

Conformational changes in the alpha- and beta-subunits of the insulin receptor identified by anti-peptide antibodies

The structure of the insulin receptor was studied with polyclonal antibodies obtained from rabbits which were immunized with synthetic peptides having a sequence identity to three regions of the alpha-subunit and five regions of the beta-subunit. None of the alpha-subunit antibodies including alpha-Pep8 (residues 40-49 (Ullrich, A., Bell, J.R., Chen, E.Y., Herrera, R., Petruzzelli, L.M., Dull, T.J., Gray, A., Coussens, L., Liao, Y.-C., Tsubokawa, M., Mason, A., Seeburg, P.H., Grunfeld, C., Rosen, O.M., and Ramachandran, J. (1985) Nature 313, 756-761), alpha-Pep7 (12 amino acid C-terminal extension (Ebina, Y., Ellis, L., Jarnagin, K., Ederly, M., Graf, L., Clauser, E., Ou, J.-H., Masiar, F., Kan, Y.W., Goldfine, I.D., Roth, R.A., and Rutter, W.J. (1985) Cell 313, 747-758], or alpha-Pep6 (residues 1-7, 9) immunoprecipitated the human insulin receptor solubilized from IM-9 lymphocytes; however, alpha-Pep8 immunoprecipitated the dithiothreitol-reduced receptor. Antibodies prepared against the N terminus of the beta-subunit (alpha-Pep5, residues 780-790) and the ATP binding site (alpha-Pep3, residues 1013-1022) did not react with the intact receptor under any conditions; however, antibodies to the C terminus of the beta-subunit (alpha-Pep1, residues 1314-1324) and to the juxta-membrane region (alpha-Pep3, residues 952-962) immunoprecipitated the solubilized receptor in both its phosphorylated and nonphosphorylated forms. In contrast, the antibody reactive with the regulatory region of the beta-subunit which contains the major autophosphorylation sites (alpha-Pep2, residues 1143-1154) only precipitated the phosphorylated form. Thus the conformation of the extracellular domain of the receptor is rigid and stabilized by disulfide bonds, whereas several regions of the intracellular domain are accessible to antibodies and undergo conformational changes during autophosphorylation.     

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.P. (1983) EMBO J. 2, 381-387). Here we describe a further significant step toward renaturation of the alpha-subunit as judged by toxin and monoclonal antibody binding. Purified T. marmorata receptor subunits were diluted with 1% lipids (asolectin) plus 0.5% Na+ cholate. An anion-exchange resin eliminated most of the detergents, leaving approximately 0.1% Na+ cholate and the lipids. After this treatment, about 20% of the alpha-subunit recovered (but not the beta-, gamma-, or delta-subunit) exhibited a high affinity for radioiodinated alpha-bungarotoxin with a KD approximately 0.5 nM. The 34,000- and 27,000-dalton proteolytic peptides of the alpha-subunit conserved this lipid-dependent toxin binding. Unlabeled alpha-toxins, hexamethonium, and carbamylcholine competed with alpha-bungarotoxin for the renatured alpha-subunit. Noncompetitive channel blockers doubled the lipid-dependent toxin-binding capacity of the alpha-subunit but had no effect on the 27,000-dalton peptide. The binding of several monoclonal antibodies to the main immunogenic region (which is particularly sensitive to denaturation) significantly increased. In particular, binding of antibody 16 changed from 1% to denatured to 100% to the lipid-renaturated alpha-subunit. The binding of these antibodies was lost with the lipid-renatured 34,000- and 27,000-dalton peptides.     

Heterogeneity of antibodies blocking the binding of bungarotoxin to the acetylcholine receptor in myasthenia

Inhibitory effect of myasthenic patients antibodies on alpha-bungarotoxin binding to the human acetylcholine receptor has been demonstrated by radioimmunoassay. By using decamethonium, an acetylcholine agonist, we have shown the existence of two antibody sub-groups reacting with the toxin-binding site: one sub-group is represented by antibodies which block the binding directly, the other by antibodies that inhibit the binding, only in the presence of decamethonium.     

Antibodies against preselected peptides to map functional sites on the acetylcholine receptor

Rabbit immune sera and mouse monoclonal antibodies were raised against the synthetic peptide Tyr-Cys-Glu-Ile-Ile-Val matching in sequence residues 127-132 of the alpha-subunit of all nicotinic acetylcholine receptors sequenced so far. Representative cholinergic ligands did not interfere with the binding of these antibodies to the receptor from Torpedo marmorata, indicating that this sequence is not part of the binding sites for cholinergic ligands. The applicability of antigenic sites analysis to the mapping of functional sites on receptor proteins is discussed.     

Location in Muscarinic Acetylcholine Receptors of Sites for [3H]Propylbenzilcholine Mustard Binding and for Phosphorylation with Protein Kinase C

Muscarinic acetylcholine receptors purified from porcine cerebra or atria were covalently labeled with [3H]propylbenzilylcholine mustard ([3H]PrBCM), and then the labeled receptors were subjected to limited hydrolysis with trypsin, V8 protease, and lysyl endopeptidase, followed by analysis involving sodium dodecyl sulfate-polyacrylamide gel electrophoresis, fluorography, autoradiography, or immunostaining. The labeled peptides were located on the basis of their reactivity with antibodies raised against three synthetic peptides with partial sequences of the m1 or m2 receptor, and of their sensitivity to endoglycosidase F, which was taken as evidence that they contain glycosylation sites near the N terminus. The [3H]PrBCM-binding site in both cerebral and atrial receptors was found to be located between the N terminus and the second intracellular loop, because the size of the smallest deglycosylated peptide that contained both the [3H]PrBCM-binding and glycosylation sites was approximately 16 kDa. Cerebral receptors were 32P-phosphorylated with protein kinase C, and the major phosphorylation sites in cerebral muscarinic receptors were found to be located in a C-terminal segment including a part of the third intracellular loop, because a 32P-labeled peptide of 12-14 kDa reacted with anti-(m1 C-terminal peptide) antiserum. The presence of an intramolecular disulfide bond, probably between Cys 98 and Cys 178 in the first and second extracellular loops, respectively, was suggested by the finding that a peptide of approximately 17 kDa containing the [3H]PrBCM-binding site, but not the glycosylation sites, was partly converted to a peptide of approximately 12 kDa on treatment with beta-mercaptoethanol.     

Antipeptide antibodies of predetermined specificity recognize and neutralize the bioactivity of the pan-specific hemopoietin interleukin 3

Ten peptides that corresponded to portions of the T cell lymphokine pan-specific hemopoietin interleukin 3 (IL 3) were synthesized, coupled to keyhole limpet hemocyanin, and used to raise antipeptide antibodies in rabbits. These antisera reacted to varying degrees with native biologically active IL 3. Antibodies directed against peptides corresponding to residues 1-29 at the NH2 terminus, 123-140 at the COOH terminus, and to residues 64-82 and 91-112 were affinity-purified on peptide columns. Immunoabsorbent columns produced from affinity-purified antibodies to the 1-29, 91-112, and 123-140 although not the 64-82 peptide were effective in depleting biologically active IL 3 from conditioned medium. However, the antibodies specific for peptides 91-112 and 123-140 had only a low affinity for native IL 3 and it was only in the case of the anti-1-29 antibodies that a significant amount of IL 3 remained bound after extensive washing and could be recovered from the column by acid elution. The affinity-purified antibodies directed to peptides 1-29, 91-112, and 123-140 significantly inhibited the biological activity of IL 3, although with different dose-response characteristics. Anti-1-29 antibodies inhibited bioactivity over a wide range of concentrations (down to 20 ng/ml) although the inhibition was never complete. In contrast, the anti-91-112 antibodies, although effective only at high concentrations, produced complete inhibition of biological activity. These experiments demonstrated that antibodies to defined peptides can be used to generate antibodies to native IL 3 and should form useful tools in analyzing the structure and function of the native molecules.