Identification and purification of a novel receptor for secretory phospholipase A(2) in porcine cerebral cortex.

A specific phospholipase A(2) receptor from porcine cerebral cortex has been characterized (K(d) = 145 nM, B(max) = 0.4 pmol/mg membrane protein) by using a radioiodinated derivative of ammodytoxin C (AtxC), a snake venom presynaptically neurotoxic group IIA phospholipase A(2). After the receptor was solubilized in a ligand-binding form, it was approximately 14,000-fold enriched by chromatography on wheat germ lectin-Sepharose and AtxC-Affi-Gel 10. The receptor is a single chain glycoprotein with an apparent molecular mass of 180 kDa and binds toxic and non-toxic phospholipases A(2) of either group I or II. It also recognizes conjugates of bovine serum albumin with mannose, N-acetylglucosamine, and galactose. In its molecular mass and pharmacological profile, the AtxC receptor resembles the M-type receptor for secretory phospholipases A(2) from rabbit skeletal muscle (a C-type multilectin, homologous to macrophage mannose receptor), yet in terms of relative abundance in brain and antigenicity, these two receptors are completely different. A further AtxC receptor of approximately 200 kDa discovered in porcine liver was, however, recognized by anti-rabbit M-type phospholipase A(2) receptor antibodies. There are, therefore, two immunologically distinct secretory phospholipase A(2) receptors of about 200 kDa in the same species. Although the liver receptor is related to the M-type secretory phospholipase A(2) receptors, the brain receptor is not and belongs to a novel group of secretory phospholipase A(2) receptors.

Identification of a new high-affinity binding protein for neurotoxic phospholipases A2.

Ammodytoxin C is a neurotoxic phospholipase A2 which blocks the release of neurotransmitter from the nerve terminal. Using a radioiodinated derivative of the toxin, we located its specific high-affinity binding site in the demyelinated P2 fraction of porcine cerebral cortex (Kd = 15 nM; Bmax = 1.5 pmol/mg membrane protein). In cross-linking experiments on a membrane preparation, 125I-ammodytoxin C labeled a protein of 25 kDa. The formation of a specific adduct was not inhibited by nontoxic phospholipases A2 or even by neurotoxic phospholipases A2 which have practically identical pathophysiological activities to ammodytoxin C: agkistrodotoxin, Oxyuranus scutellatus 2 phospholipase A2, taipoxin, beta-bungarotoxin, notexin, and crotoxin. 125I-ammodytoxin C specific cross-linking was inhibited, however, by mannosylated BSA, suggesting the presence of a carbohydrate-recognition domain in the acceptor structure. According to the pharmacological and structural properties, the ammodytoxin acceptor from porcine cerebral cortex differs from other so far identified as phospholipase A2 acceptors and represents a new type of a high-affinity binding protein for neurotoxic phospholipases A2.

Ammodytin L, an inactive phospholipase A2 homologue with myotoxicity in mice, binds to the presynaptic acceptor of the beta-neurotoxic ammodytoxin C in Torpedo: an indication for a phospholipase A2 activity-independent mechanism of action of beta-neurotoxins in fish?

A Ser48 phospholipase A2-homologue, ammodytin L, which is myotoxic in mammals and devoid of any phospholipase A2 activity, completely inhibits the specific binding of the neurotoxic phospholipase A2, ammodytoxin C, to fish presynaptic membranes from Torpedo marmorata electric organ. In cross-linking experiments, 125I-ammodytin L labels the same membrane proteins as 125I-ammodytoxin C (70, 38.5-57.4 and 19.7 kDa). The formation of these adducts is completely prevented by the presence of ammodytoxin C but not of a non-toxic phospholipase A2, ammodytin I2. A chimeric phospholipase A2, constructed by associating the N-terminal half of ammodytoxin to the C-terminal half of ammodytin L, possesses a low, but significant phospholipase A2 activity, however it is not toxic to mice, probably due to abolition of the specific neuronal acceptor binding in mammals. Nevertheless, the chimeric phospholipase A2 is able to interact with the ammodytoxin acceptor in Torpedo marmorata electric organ. The existence of neuronal acceptors for ammodytin L and for the chimeric phospholipase A2 suggests that they may act as neurotoxins in fish. As ammodytin L does not possess any enzymatic activity it, therefore, appears to be an excellent tool to investigate the mechanism of action of beta-neurotoxins independently of their phospholipase A2 activity.