The action of botulinum toxin on cholinergic nerve terminals isolated from the electric organ of Torpedo marmorata. Detection of a putative toxin receptor

Botulinum neurotoxin type A (BoNTx) inhibits the release of acetylcholine (ACh) from Torpedo electric organ synaptosomes. We have studied several biochemical and morphological aspects in order to characterize the molecular interactions of BoNTx intoxication in our preparation. 1. We are describing for the first time an electrophoretic band from cholinergic presynaptic plasma membrane (PSPM) that is recognized by 125I-BoNTx as a putative BoNTx receptor. 2. Furthermore we describe direct interaction of botulinum toxin-gold complexes with synaptic vesicles through the three-step model of the BoNTx intoxication.     

300-kD subsynaptic protein copurifies with acetylcholine receptor-rich membranes and is concentrated at neuromuscular synapses

Acetylcholine receptor-rich membranes from the electric organ of Torpedo californica are enriched in the four different subunits of the acetylcholine receptor and in two peripheral membrane proteins at 43 and 300 kD. We produced monoclonal antibodies against the 300-kD protein and have used these antibodies to determine the location of the protein, both in the electric organ and in skeletal muscle. Antibodies to the 300-kD protein were characterized by Western blots, binding assays to isolated membranes, and immunofluorescence on tissue. In Torpedo electric organ, antibodies to the 300-kD protein stain only the innervated face of the electrocytes. The 300-kD protein is on the intracellular surface of the postsynaptic membrane, since antibodies to the 300-kD protein bind more efficiently to saponin-permeabilized, right side out membranes than to intact membranes. Some antibodies against the Torpedo 300-kD protein cross-react with amphibian and mammalian neuromuscular synapses, and the cross-reacting protein is also highly concentrated on the intracellular surface of the post-synaptic membrane.     

Binding of a Glycera convoluta neurotoxin to cholinergic nerve terminals triggers a Ca-dependent acetylcholine release

The venom glands of the annelid Glycera convoluta contain a neurotoxin which triggers ACh release from frog motor terminals and Torpedo synaptosomes. This neurotoxin binds to presynaptic, but not postsynaptic plasma membranes prepared from Torpedo electric organ. The binding site is an ectocellularly oriented protein. The binding does not require Ca. It is inhibited by pretreatment of the membrane by Concanavalin A. The toxin induced ACh release is Ca-dependent and inhibited by D 600.     

Detection with Monoclonal Antibodies of a 15-kDa Proteolipid in Both Presynaptic Plasma Membranes and Synaptic Vesicles in Torpedo Electric Organ

A protein, the mediatophore, has been purified from Torpedo electric organ presynaptic plasma membranes. This protein mediates the release of acetylcholine through artificial membranes when activated by calcium and is made up of 15-kDa proteolipid subunits. After immunization with purified delipidated mediatophore, monoclonal antibodies binding to the 15-kDa proteolipid band on Western blots of purified mediatophore were selected. A 15-kDa proteolipid antigen was also detected in cholinergic synaptic vesicles. Using an immunological assay, it was estimated that presynaptic plasma membranes and synaptic vesicles contain similar proportions of 15-kDa proteolipid antigen. Detection by immunofluorescence in the electric organ showed that only nerve endings were labeled. In electric lobes, the staining was associated with intracellular membranes of the electroneuron cell bodies and in axons. Nerve endings at Torpedo neuromuscular junctions were also labeled with anti-15-kDa proteolipid monoclonal antibodies.     

Dystrophin in electric organ of Torpedo californica homologous to that in human muscle

We have found that dystrophin is highly concentrated at neuromuscular junctions and innervated membranes of the electric organ of Torpedo californica. In acetylcholine receptor-rich Torpedo membrane preparations dystrophin represents approximately 0.4% of total protein and can be extracted from these membranes by alkaline treatment in the absence of detergent, indicating that it is a peripheral membrane protein. Polyclonal antibodies raised against electrophoretically isolated Torpedo dystrophin cross-react with dystrophin in human muscle and unequivocally discriminate between normal and Duchenne muscular dystrophy patient's muscle. These results indicate that dystrophin is phylogenetically a highly conserved protein and that the relatively abundant dystrophin in electric organ would facilitate further investigations of its structure and function.     

Binding of a Glycera convoluta neurotoxin to cholinergic nerve terminal plasma membranes

The crude extract of venom glands of the polychaete annelid Glycera convoluta triggers a large Ca2+-dependent acetylcholine release from both frog motor nerve terminals and Torpedo electric organ synaptosomes. This extract was partially purified by Concanavalin A affinity chromatography. The biological activity was correlated in both preparations to a 300,000-dalton band, as shown by gel electrophoresis. This confirmed previous determinations obtained with chromatographic methods. This glycoprotein binds to presynaptic but not postsynaptic plasma membranes isolated from Torpedo electric organ. Pretreatment of intact synaptosomes by pronase abolished both the binding and the venom- induced acetylcholine release without impairing the high K+-induced acetylcholine release. Pretreatment of nerve terminal membranes by Concanavalin A similarly prevented the binding and the biological response. Binding to Torpedo membranes was still observed in the presence of EGTA. An antiserum directed to venom glycoproteins inhibited the neurotoxin so we could directly follow its binding to the presynaptic membrane. Glycera convoluta neurotoxin has to bind to a ectocellularly oriented protein of the presynaptic terminal to induce transmitter release.     

Isolation of a cDNA clone for a catalytic subunit of Torpedo marmorata acetylcholinesterase

We have constructed a cDNA library from Torpedo marmorata electric organ poly(A+) RNA in the lambda phage expression vector lambda gt11. This library has been screened with polyclonal anti-acetylcholinesterase antibodies. One clone, lambda AChE1, produced a fusion protein which was recognized by the antibodies and which prevented the binding of native acetylcholinesterase in an enzymatic immune assay. These results indicate that lambda AChE1 contains a cDNA insert coding for a part of a catalytic subunit of Torpedo acetylcholinesterase. The 200-base-pair cDNA insert hybridized to three mRNAs (14.5, 10.5 and 5.5 kb) from Torpedo electric organs. These mRNAs were also detected in Torpedo electric lobes.     

The putative agrin receptor binds ligand in a calcium-dependent manner and aggregates during agrin-induced acetylcholine receptor clustering.

Agrin derived from Torpedo electric organ induces the clustering of acetylcholine receptors (AChRs) on cultured myotubes. As a first step toward characterizing the plasma membrane receptor for agrin, we have examined agrin binding to cultured myotubes. Agrin binding is saturable as measured by radioimmunoassay and, like agrin-induced AChR clustering, requires extracellular calcium. Immunofluorescence shows that on myotubes incubated with agrin at 4 degrees C, agrin binds in a uniform, finely punctate pattern that correlates poorly with the distribution of AChRs. Myotubes stimulated with agrin at 37 degrees C for greater than or equal to 2 hr show a coclustering of agrin binding sites and AChRs. By contrast, if anti-AChR antibodies are used either to cluster or to internalize AChRs, the distribution and number of agrin binding sites remain unchanged. The aggregation and calcium dependence of the putative agrin receptor may represent important control points in postsynaptic differentiation.     

Antipeptide Antibodies Against a Torpedo Cysteine-String Protein

Abstract: An antipeptide antiserum was raised against the C-terminal undecapeptide of a Torpedo cysteine-string protein (csp), a putative subunit or modulator of presynaptic calcium channels. This antiserum was shown to identify selectively the 27-kDa in vitro translation product of the csp cRNA both by immunoprecipitation and on immunoblots. When affinity-purified anti-csp antibodies were used to probe immunoblots of membrane proteins from Torpedo electric organ or liver, specific immunoreactivity was detected only in electric organ. This immunoreactivity was associated principally with a single protein species of about 34 kDa. These results indicate that csp immunoreactivity is detectably expressed in electroplax, a heavily innervated tissue, but not in liver, which should have an appreciably lower abundance of presynaptic calcium channel proteins. Moreover, the increased relative molecular mass of csp in electric organ (compared with in vitro translated material) implies that csp is posttranslationally modified. Finally, immunoblot analysis of either intact, alkali-treated, or solubilized membrane fractions of electric organ reveals that csp is predominantly a membrane protein.     

Binding of antibodies to acetylcholine receptors in Electrophorus and Torpedo electroplax membranes

Antisera against purified acetylcholine receptors from the electric tissues of Torpedo californica and of Electrophorus electricus were raised in rabbits. The antisera contain antibodies which bind to both autologous and heterologous receptors in solution as shown by an immunoprecipitation assay. Antibodies in both types of antisera bind specifically to the postjunctional membrane on the innervated surface of the intact electroplax from Electrophorus electric tissue as demonstrated by an indirect immunohistochemical procedure using horseradish peroxidase conjugated to anti-rabbit IgG. Only anti-Electrophorus receptor antisera, however, cause inhibition of the receptor-mediated depolarization of the intact Electrophorus electroplax. The lack of inhibition by anti-Torpedo receptor antibodies, which do bind, suggests that the receptor does not undergo extensive movement during activity. The binding of anti-Torpedo antibodies to receptor-rich vesicles prepared by subcellular fractionation of Torpedo electric tissue was demonstrated by both direct and indirect immunohistochemical methods using ferritin conjugates. These vesicles can be conveniently collected and prepared for electron microscopy on Millipore filters, a procedure requiring only 25 micrograms of membrane protein per filter. In addition, it was possible to visualize the binding of anti-Torpedo receptor antibodies directly, without ferritin. These anti-Torpedo receptor antibodies, however, do not inhibit the binding of acetylcholine or of alpha-neurotoxin to receptor in Torpedo microsacs but do inhibit binding of alpha-neurotoxin to Torpedo receptor in Triton X-100 solution. It is likely that the principal antigenic determinants on receptor are at sites other than the acetylcholine-binding sites and that inhibition of receptor function, when it occurs, may be due to a stabilization by antibody binding of an inactive conformational state.     

α-TOXIN BINDING PROTEINS IN THE ELECTRIC ORGAN OF TORPEDO MARMORATA STUDIED BY IMMUNOCHEMICAL METHODS

—Crossed immunoelectrophoretic techniques were developed to study the efficiency of the various purification steps in the isolation of nicotinic acetylcholine receptor (nAChR) from Torpedo mormorata electric organ. A new α-neurotoxin binding assay based on immunoelectrophoresis is also presented. In crude extracts of Torpedo electric organ membranes one type of receptor molecule (M ñ; 300 , 000) was found; an earlier described higher molecular form was shown to be an artifact of affinity chromatography. Polyvalent antibodies against Torpedo electroplaque membranes, antibodies against purified membrane proteins and against Naja naja siamensisα-neurotoxin revealed four α-neurotoxin binding antigens (including nAChR). Two of these, nAChR and T₂, were specific for electroplaque membrane and showed partial immunoidentity but different biochemical and physical properties.     

Antigenic role of single residues within the main immunogenic region of the nicotinic acetylcholine receptor.

The target of most of the autoantibodies against the acetylcholine receptor (AChR) in myasthenic sera is the main immunogenic region (MIR) on the extracellular side of the AChR alpha-subunit. Binding of anti-MIR monoclonal antibodies (mAbs) has been recently localized between residues alpha 67 and alpha 76 of Torpedo californica electric organ (WNPADYGGIK) and human muscle (WNPDDYGGVK) AChR. In order to evaluate the contribution of each residue to the antigenicity of the MIR, we synthesized peptides corresponding to residues alpha 67-76 from Torpedo and human AChRs, together with 13 peptide analogues. Nine of these analogues had one residue of the Torpedo decapeptide replaced by L-alanine, three had a structure which was intermediate between those of the Torpedo and human alpha 67-76 decapeptides, and one had D-alanine in position 73. Binding studies employing six anti-MIR mAbs and all 15 peptides revealed that some residues (Asn68 and Asp71) are indispensable for binding by all mAbs tested, whereas others are important only for binding by some mAbs. Antibody binding was mainly restricted to residues alpha 68-74, the most critical sequence being alpha 68-71. Fish electric organ and human MIR form two distinct groups of strongly overlapping epitopes. Some peptide analogues enhanced mAb binding compared with Torpedo and human peptides, suggesting that the construction of a very antigenic MIR is feasible.     

Identification of a 43-kDa polypeptide associated with acetylcholine receptor-enriched membranes as MM creatine kinase

Creatine kinase isoenzymes from Torpedo californica electric organ, skeletal muscle, and brain were purified and characterized. Torpedo electric organ and skeletal muscle creatine kinase have identical apparent Mr, electrophoretic mobility, and cyanogen bromide fragments. The electrophoretic mobility of the Torpedo creatine kinase was anodal as compared to mammalian MM creatine kinase. No creatine kinase isoenzyme with an electrophoretic mobility similar to mammalian BB creatine kinase was seen in any of the Torpedo tissues examined. Hybridization studies demonstrate the Torpedo electric organ creatine kinase to be composed of identical subunits and capable of producing an enzymatically active heterodimer when combined with canine BB creatine kinase. Creatine kinase from sucrose gradient-purified Torpedo electric organ acetylcholine receptor-rich membranes has an electrophoretic mobility identical with the cytoplasmic isoenzyme and an apparent Mr identical with mammalian MM creatine kinase. Western blot analysis showed Torpedo electric organ skeletal muscle creatine kinase and acetylcholine receptor-enriched membrane creatine kinase reacted with antiserum specific for canine MM creatine kinase. NH2-terminal amino acid sequence determinations show considerable sequence homology between human MM, Torpedo electric organ, chicken MM, and porcine MM creatine kinase. The acetylcholine receptor-associated creatine kinase is, therefore, identical with the cytoplasmic form from the electric organ and is composed of M-subunits.     

ASSOCIATION OF SYNTAXIN WITH SNAP 25 AND VAMP (SYNAPTOBREVIN) IN TORPEDO SYNAPTOSOMES

Two proteins of the presynaptic plasma membrane, syntaxin and SNAP 25, and VAMP/synaptobrevin, a synaptic vesicle membrane protein, form stable protein complexes which are involved in the docking and fusion of synaptic vesicles at the mammalian brain presynaptic membrane. Similar protein complexes were revealed in an homogeneous population of cholinergic synaptosomes purified from Torpedo electric organ by combining velocity sedimentation and immunoprecipitation experiments. After CHAPS solubilization, virtually all the nerve terminal syntaxin was found in the form of large 16 S complexes, in association with 65% of SNAP 25 and 15% of VAMP. Upon Triton X100 solubilization, syntaxin was still recovered in association with SNAP 25 and VAMP but in smaller 8 S complexes. A small (2–5%) percentage of the nerve terminal 15 kDa proteolipid subunit of the v-H⁺ ATPase and of mediatophore was copurified with syntaxin, using two different antisyntaxin monoclonal antibodies. The use of an homogeneous population of peripheral cholinergic nerve terminals allowed us to extend results on the composition of the brain presynaptic protein complexes to the Torpedo electric organ synapse, a model of the rapid neuromuscular synapses. Copyright © 1996 Elsevier Science Ltd     

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.     

Monoclonal Antibody Tor 23 Recognizes a Determinant of a Presynaptic Acetylcholinesterase

A significant proportion of the acetylcholinesterase that is present in the electric organ of Torpedo californica exists as a presynaptic membrane molecule. The monoclonal antibody Tor 23 binds the Torpedo presynaptic nerve membrane where it recognizes a polypeptide of 68,000 daltons. Our present studies indicate that Tor 23 identifies acetylcholinesterase. From the homogenates of Torpedo nerve terminals, Tor 23 immunoprecipitates measurable esterase activity. Esterase precipitation was not observed with no Tor 23 added; nor was it observed with any other test antibodies, including other Tor antibodies, in particular, Tor 70, which binds, as does Tor 23, to the presynaptic nerve membrane. The esterase activity was specific for acetylcholinesterase. Our studies indicate the molecule defined by Tor 23 has the solubility properties described for that of presynaptic acetylcholinesterase: it is soluble in detergent-treated electroplax homogenates and insoluble in high-salt extractions. In sections of Torpedo back muscle, both nerve and endplate acetylcholinesterase can be detected histochemically. Tor 23 localizes to the nerve and is not clustered at the endplate. The utility of the antibody Tor 23 thus includes biochemical and histological analyses of the multiple forms of acetylcholinesterase.     

Characterization of a membrane protein from cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata

Rabbits were immunized with cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata. The resultant antiserum had one major antibody activity against an antigen called the Torpedo vesicle antigen. This antigen could not be demonstrated in muscle, liver or blood and is therefore, suggested to be nervous-tissue specific. The vesicle antigen was quantified in various parts of the nervous system and in subcellular fractions of the electric organ of Torpedo marmorata and was found to be highly enriched in synaptic vesicle membranes. The antigen bound to concanavalin A, thereby demonstrating the presence of a carbohydrate moiety. By means of charge-shift electrophoresis, amphiphilicity was demonstrated, indicating that the Torpedo vesicle antigen is an intrinsic membrane protein. The antigen was immunochemically unrelated to other brain specific proteins such as 14-3-2, S-100, the glial fibrillary acidic protein and synaptin. Furthermore, it was unrelated to two other membrane proteins, the nicotinic acetylcholine receptor and acetylcholinesterase, present in Torpedo electric organ. The antiserum against Torpedo synaptic vesicles did not react with preparations of rat brain synaptic vesicles or ox adrenal medullary chromaffin granules.     

Localization of dystrophin relative to acetylcholine receptor domains in electric tissue and adult and cultured skeletal muscle

Two high-affinity mAbs were prepared against Torpedo dystrophin, an electric organ protein that is closely similar to human dystrophin, the gene product of the Duchenne muscular dystrophy locus. The antibodies were used to localize dystrophin relative to acetylcholine receptors (AChR) in electric organ and in skeletal muscle, and to show identity between Torpedo dystrophin and the previously described 270/300-kD Torpedo postsynaptic protein. Dystrophin was found in both AChR-rich and AChR-poor regions of the innervated face of the electroplaque. Immunogold experiments showed that AChR and dystrophin were closely intermingled in the AChR domains. In contrast, dystrophin appeared to be absent from many or all AChR-rich domains of the rat neuromuscular junction and of AChR clusters in cultured muscle (Xenopus laevis). It was present, however, in the immediately surrounding membrane (deep regions of the junctional folds, membrane domains interdigitating with and surrounding AChR domains within clusters). These results suggest that dystrophin may have a role in organization of AChR in electric tissue. Dystrophin is not, however, an obligatory component of AChR domains in muscle and, at the neuromuscular junction, its roles may be more related to organization of the junctional folds.     

A protein homologous to the Torpedo postsynaptic 58K protein is present at the myotendinous junction

The 58K protein is a peripheral membrane protein enriched in the acetylcholine receptor (AChR)-rich postsynaptic membrane of Torpedo electric organ. Because of its coexistence with AChRs in the postsynaptic membrane in both electrocytes and skeletal muscle, it is thought to be involved in the formation and maintenance of AChR clusters. Using an mAb against the 58K protein of Torpedo electric organ, we have identified a single protein band in SDS-PAGE analysis of Xenopus myotomal muscle with an apparent molecular mass of 48 kD. With this antibody, the distribution of this protein was examined in the myotomal muscle fibers with immunofluorescence techniques. We found that the 48K protein is concentrated at the myotendinous junctions (MTJs) of these muscle fibers. The MTJ is also enriched in talin and vinculin. By double labeling muscle fibers with antibodies against talin and the 48K protein, these two proteins were found to colocalize at the membrane invaginations of the MTJ. In cultured myotomal muscle cells, the 48K protein and talin are also colocalized at sites of membrane-myofibril interaction. The 48K protein is, however, not found at focal adhesion sites in nonmuscle cells, which are enriched in talin. These data suggest that the 48K protein is specifically involved in the interaction of myofibrillar actin filaments with the plasma membrane at the MTJ. In addition to the MTJ localization, 48K protein is also present at AChR clusters both in vivo and in vitro. Thus, this protein is shared by both the MTJ and the neuromuscular junction.     

Association of acetylcholinesterase with the external surface of the presynaptic plasma membrane in Torpedo electric organ

A high acetylcholinesterase (AChE) activity was found associated with pure cholinergic synaptosomes prepared from Torpedo electric organ. This activity was bound to the presynaptic plasma membrane upon subfractionation on sucrose density gradients. It was not solubilized in the presence of 2 M MgCl₂ but in the presence of Triton X 100. This presynaptic AChE activity corresponded to a hydrophobic form of the enzyme with a sedimentation coefficient of 5.5 S in our conditions. More than 80% of the AChE activity of intact synaptosomes was externally oriented. The presynaptic AChE activity could represent as much as 25% of the total activity in Torpedo electric organ.