The primary structure of ammodytin L, a myotoxic phospholipase A₂ homologue from Vipera ammodytes venom

Abstract

A new myotoxic phospholipase Az homologue, having a serine residue in position 49 instead of highly conserved aspartic acid, was found in the venom of Vipevu ummodytes. The primary structure revealed additional mutations in the positions important for enzymatic activity. Tyr28 is exchanged for a histidine and Gly33 for asparagine. These changes render earlier-reported weak enzymatic activity unlikely. The role of this rather abundant venom fraction is apparently in myotoxicity, which was confirmed in the muscle-cell culture from neonatal rats. The muscle-cell culture proved to be a good tool to investigate the effects of various myotoxins on muscle cells. MATERIALS AND METHODS Protein sequencingAmmodytin L, formerly designated as venom fraction 1, was isolated as described [lo, 111. For the primary structure determination, Applied Biosystems gas-phase sequencer, model470A, connected on-line to a model 120A phenylthiohydantoin (PTH)-amino acid analyser was used. Cys residues were alkylated with radioactive [3H]iodoacetic acid (Amersham) and determined as cdrboxymethyl-PTH derivatives on reverse-phase HPLC and by radioactive detection on a Beckmann LSlOOC fl-counter. 1-Chloro-3-tosylamido-4-phenyl-butan-2-one-treated (TPCK) trypsin was obtained from Sigma. Endoproteinase Lys-C was from Boehringer. Hydroxylamine hydrochloride from Fluka was used for AsnGly cleavage. All other chemicals were of sequential grade. Amino acid analyses of peptide hydrolysates, obtained with 6 M HCl at 13 0" C for 24 h, were performed by HPLC, using an ODS 2 column and pre-column derivatisation with opht ha1 aldehyde.Carboxymethylated ammodytin L was maleylated according to the procedure of Butler and Hartley [13]. Trypticckw age of maleylated carboxymethylated ammodytin L was performed at 37°C and pH 8.2 in 0.5 M N-methylmorpholinel acetate with 2% (by mass) proteinase for 40 min. Cleavage with 2% (by mass) endoproteinase Lys-C was performed at 38°C and pH 8.1 in 0.25 M ammonium hydrogencarbonate for 6 h. Hydroxylamine cleavage of the Asn-Gly bond was performed according to Bornstein and Balian [14]. Peptide purification was performed by Sephacryl S-200 gel-filtration chromatography (1 50 cm x 0.5 cm) using a flow rate of 2.4 ml/ h with 50% HCOOH as the eluent and by HPLC on Chrompack reverse-phase C8 and CIS columns (100 mm x 3 mm) equilibrated with 0.1 % trifluoroacetic acid in water and eluted by linear gradients to 80% acetonitrile and 0.1% trifluoroacetic acid. The flow rate was 1 ml/min. The absorbance was monitored either at 277 nm or 215 nm.

Secreted phospholipase A2-induced neurotoxicity and epileptic seizures after intracerebral administration: An unexplained heterogeneity as emphasized with paradoxin and crotoxin

“…For the former, E280, 18,117 M -1 cm -1 and Mr 13,936 were used. For the latter E280 was 13,211 M -1 cm -1 (Krizaj, personal communication) and Mr 13,868 (Krizaj et al, 1991). All the toxins and enzymes were dissolved in sterile normal saline (NaCl 0.15 M), aliquoted and kept frozen.…”

“…To explore the involvement of the catalytic activity, we evaluated the epileptogenic properties of alkylated CB using the widely used inhibitor pbromophenacyl bromide (Chang and Su, 1982;Délot and Bon, 1992) and of ammodytin L (AML), a Ser 49 phospholipase A 2 homologue of ATX A (Krizaj et al, 1991) that is devoid of enzymatic activity (Rufini et al, 1992) and peripheral neurotoxicity, while surprisingly being able to compete with neurotoxic ammodytoxins in Torpedo synaptosomes (Pungercar et al, 1998) but very little in bovine brain cortex (Krizaj et al, 1995). To our knowledge, this is the first detailed report on central toxicity of PDX, on acute central neurotoxic lesions after sPLA 2 s injection and the first to emphasize the large heterogeneity in sPLA 2 s epileptogenic properties.…”

Differential susceptibility of C2C12 myoblasts and myotubes to group II phospholipase A2 myotoxins from crotalid snake venoms

“…Studies using a variety of cell lines in culture have shown that myotoxic group I PLA 2 s are not cytolytic, whereas group II PLA 2 s exert a broad cytolytic activity, 7,[13][14][15][16][17][18] which correlates with their in vivo myotoxic action. 19 Several of the group II myotoxins studied in cell culture models belong to the subgroup of Lys49 PLA 2 homologues, protein variants that are enzymically inactive due to a number of amino acid substitutions, including the critical Asp49 to Lys49 change (reviewed by Lomonte et al 12 ).…”

Differential gene expression profiles in the venom gland/sac of Orancistrocerus drewseni (Hymenoptera: Eumenidae)

“…The basic function of PLA2 is the hydrolysis of phospholipids to lysophospholipids and free fatty acids using Ca 21 as a co-factor (Six and Dennis, 2000). In addition, some catalytic venom PLA2s are known to have other physiological functions, including presynaptic and postsynaptic neurotoxicity (Bon et al, 1979), myotoxicity (Krizaj et al, 1991;Mebs and Samejima, 1986;Ward et al, 1998), hemolytic activity (Costa and Palma, 2000;de Oliveira and Palma, 1998;Ho and Ko, 1988), anticoagulant activity (Andriao-Escarso et al, 2002;Jabeen et al, 2005), and antibacterial activity (Nevalainen et al, 2008). Secretory PLA2s typically have 5-8 intramolecular disulfide bridges and a highly conserved active site and Ca 21 -binding loop (Scott et al, 1990a,b;Verheij et al, 1980;White et al, 1990).…”