Binding proteins on synaptic membranes for crotoxin and taipoxin, two phospholipases A 2 with neurotoxicity

Abstract

Crotoxin and taipoxin are both neurotoxic phospholipases A 2 capable of affecting the presynaptic activity to bring about ultimate blockade of synaptic transmission. The enzymatic activity has generally been considered to be necessary but not sufficient for the blockade. Since many phospholipases A 2 with comparable or even higher enzymatic activity are not toxic, it has been postulated that the difference lies in the affinity of binding to the presynaptic membrane. In confirmation of this proposition, we and others have previously shown that iodinated crotoxin and taipoxin bind specifically with high affinity to the isolated synaptic membrane fraction from guinea-pig brain, whereas specific binding is not detected with the nontoxic pancreatic phospholipase A 2. Experiments based on photoaffinity labeling and simple chemical cross-linking techniques have led to the identification of three polypeptides preferentially present in neuronal membranes as (subunits of) the binding protein(s) for crotoxin and/or taipoxin. Some, but not all, other toxic phospholipases A 2 also appear to be ligands for the three polypeptides. We now report studies on partial purification of these polypeptides using affinity chromatography and other techniques. In order to learn the normal physiological roles played by the toxin-binding proteins, the phospholipase-independent effects of the toxins on the synaptosomes have been sought. We have found that under Ca 2+-free condition, taipoxin or crotoxin inhibits with ic 50 of 20–1000 nM the Na +-dependent uptake of norepinephrine, dopamine and serotonin by the synaptosomes. In contrast, choline uptake is not affected. Furthermore, the high-affinity site for [ 3H]desipramine binding, known to be the norepinephrine transporter, is inhibited with an ic 50 of 14nM by taipoxin independent of phospholipase activity. These results are strong indications that certain synaptosomal biogenic amine transporters are part of the binding proteins for taipoxin and crotoxin. Chemical modification at Tyr-21 of the phospholipase subunit of crotoxin greatly reduces the neurotoxicity and the binding affinity with little effect on the enzymatic activity of the toxin. Hence, Tyr-21 may be an important residue for the binding of crotoxin and perhaps other phospholipase A 2 neurotoxins. Even stronger evidence comes from the finding that replacement of the corresponding Phe residue of bovine pancreatic phospholipase A 2 with Tyr by site-directed mutagenesis enables it to compete with an ic 50 of 1 μM for the binding of [ 125I]crotoxin, contrasting sharply with the complete lack of such ability with the wild type even at 50 μM. In addition, rat phospholipase A 2, which has a corresponding Tyr residue, can inhibit the binding of [ 125I]crotoxin.