Bernard W. Fulpius

Monoclonal antibodies directed against the neurotransmitter binding site of nicotinic acetylcholine receptor

The pathogenesis of the disease myasthenia gravis (MG) and its animal model experimental autoimmune myasthenia gravis (EAMG) involves an autoimmune reaction to nicotinic acetylcholine receptor (nAcChR) [l--5], where anti-nAcChR antibodies effectively impair transmission at the neuromuscular junction [ 1,6]. Hence, the immunogenic properties of nAcChR are of major consideration. The ability to differentiate the antigenic determinants of nAcChR would be of great use by permitting a itetailed analysis of the importance of different determinants in MG, as well as providing a new dimension for structural and functional studies of nAcChR. The lymphocyte hybridization procedure provides an opportunity to develop such antigenic site-specific monoclonal antibodies (mAb) [7]. We have applied the procedure to nAcChR and report the isolation, in particular, of mAbs directed: (i) at the neurotransmitter-binding site; (ii) close to the neurotransmitter-binding site. These mAbs should help delineate the contribution of this region of nAcChR to the pathogenesis of MG.

Binding properties and subclass distribution of anti-acetylcholine receptor antibodies in myasthenia gravis

Acetylcholine receptor antibodies were studied in the serum of 21 myasthenic patients. In 18 cases antibodies directed against sites other than the toxin binding were present whereas in 10 cases only there was a measurable inhibition of the ligand binding site. These 10 sera were from the 6 patients in stage IIB, III and IV and from 4 of the 12 patients in stage IIA. Antibodies against both non-toxin and ligand binding sites were measured in IgG subclasses. Most of the antibodies of the first type belonged to either subclass 1 or 3. They were, however, never absent from subclasses 2 and 4. Antibodies of the second type were not found in subclasses 2 and 4 except in one case. In 3 cases they were present exclusively in subclass 3. In 3 patients there was no correlation between the subclass distribution of the antibodies for the different binding sites.

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+ (2mM) 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 alpha-[3H]neurotoxin.

Purification from bee venom of melittin devoid of phospholipase A2 contamination.

Abstract Conditions for the inactivation of phospholipase A2 which contaminates melittin preparations were studied. A method for the purification of that peptide from bee venom is proposed. It gives, with a high recovery, a product devoid of phospholipase A2 activity. In the first step, the venom is fractionated by gel filtration. Then the phospholipase A2 still present in the melittin fraction is destroyed by sequential sulfitolysis and cyanogen bromide cleavage. This leaves the melittin intact. The final cation-exchange chromatography yields an homogeneous melittin preparation as analyzed by gel filtration, reverse-phase HPLC, and amino acid analysis.

Inactivation and solubilization of opiate receptors by phospholipases A2.

(1) As previously shown, stereospecific binding of opiates to membrane bound receptors is inhibited by treatment with small amounts of phospholipase A2 from Vipera russelli. This effect is quantified and compared with the enzymes from the venoms of Naja Naja siamensis, Apis Mellifica and from porcine pancreas. All enzymes are equally effective. The inhibition is due to partial phospholipid hydrolysis leading to inactivation of membrane-bound receptor. (2) Bee venom phospholipase A2 together with the synergistically acting peptide, melittin, causes receptor solubilization up to 80% of preformed receptor-ligand complex can be solubilized in this manner. (3) Lysophosphatidylcholine, a product of phospholipid hydrolysis, solubilizes performed receptor-ligand complex to a similar extent. Several other detergents were tested for their ability to solubilize receptor-ligand complex. Digitonin appears to be most effective in solubilizing such a complex.

Purification and chemical characterization of melittin and acetylated derivatives

Melittin, the main basic and hydrophobic peptide of bee venom, displays marked detergent-like properties. At high peptide concentration, and depending on salt and pH, it forms a tetramer. This is prevented by using urea. A purification procedure in presence of 4.0 M urea was developed to prepare melittin in its monomeric form, free of other venom constituents such as N alpha-formyl melittin, degradation products of peptides and phospholipase A2. NH2-residues on the melittin molecule were modified by reaction with acetic anhydride to alter the asymmetrical charge distribution supposed to confer detergent-like properties to the molecule. This gave rise to di- and mono acetyl derivatives which could be used, once isolated, to study further the melittin structure-activity relationship.

ACETYLCHOLINE RECEPTOR AND MYASTHENIA GRAVIS

The following evidence favors the idea that the acetylcholine receptor (AChR) might be involved in myasthenia gravis (MG) as an autoantigen. Fambrough et al. showed an 80% reduction and a change in the distribution of AChR in the neuromuscular junction of myasthenic patients.l Rabbits immunized with AChR purified from electric fishes developed a flaccid paralysis.*-G Antibodies directed against AChR were detected in the serum of myasthenic patient^.^ Lymphocytes from patients with MG were stimulated when cultured in vitro with a water-soluble AChR fraction extracted from the eel Electrophorus electricus.* Finally, a serum factor in patients with MG prevented the binding of toxin to normal human neuromuscular junctions.9 In keeping with this hypothesis, we have tried to answer the following questions: (1) If AChR is an autoantigen, why does it become so, where is it located, and how can we detect it? ( 2 ) Are the circulating anti-AChR antibodies able to penetrate the neuromuscular junction? ( 3 ) Are the experimental models of MG suitable for the necessary study of the involvement of the thymus and possible cellular-mediated immunity?

Selective solubilization by melittin of glycophorin A and acetylcholinesterase from human erythrocyte ghosts

Melittin, the main basic and hydrophobic peptide of bee venom, has been used for solubilizing membrane components of the human erythrocyte ghost. Up to 1.0 mM, it does not extract any phospholipid. Between 0.1 and 1.0 mM, it solubilizes partially glycophorin A and acetylcholinesterase. When the membrane is first degraded by phospholipase A2, the solubilization of both proteins by melittin is total, and 48% of the phospholipids are removed, mainly as lysoproducts, whereas phospholipase A2, by itself, has no solubilizing properties. In its melittin-solubilized state, acetylcholinesterase is in a dimeric form and displays a slow time-dependent irreversible inactivation. Triton X-100 at 1.0% (v/v) interrupts the inactivation. We suggest that melittin binds to the hydrophobic site of acetylcholinesterase which anchors it in the lipid bilayer.

The Role of Humoral Immunity in Myasthenia Gravis

Publisher Summary This chapter discusses the (i) evidence favoring a role of anti-AChR antibodies in the synaptic dysfunction, (ii) data concerning the specific site of action of these antibodies, and (iii) data concerning their mechanism of action in the pathogenesis of myasthenia gravis (MG). Recent data from the studies on MG and experimental autoimmune MG indicate that anti-AChR antibodies play a major role in the impairment of neuromuscular transmission. Thus, it seems conceivable that antibodies directed against synaptic receptors can interfere with the normal function of synapses. This mechanism might not be restricted to the neuromuscular junctions, but could also take place in central synapses. Recent data favor such a hypothesis in the case of MG. It is concluded that in vivo complement mediated destruction of the postsynaptic membrane consecutive to binding of anti-AChR antibodies to AChR is the major factor causing synaptic dysfunction.

Monoclonal anti-torpedo receptor antibodies used to study antibody heterogeneity in myasthenic sera

Monoclonal antibodies against Torpedo acetylcholine receptor were used to define the binding regions of cross-reacting, anti-receptor antibodies from sera of myasthenic patients. Cross-reacting antibodies were directed mainly against the toxin-binding region of the receptor and a region remote from the acetylcholine-binding site. Few patients had antibodies against the acetylcholine-binding site region. The monoclonal antibodies provide probes for defining the heterogeneity of anti-receptor antibodies in myasthenic sera.