Poneratoxin, a neurotoxin from ant venom

Abstract

Poneratoxin is a small neuropeptide found in the venom of the ant Paraponera clavata. It is stored in the venom reservoir as an inactive 25-residue peptide. Here we describe both chemically synthesized poneratoxin and poneratoxin obtained by expression in insect cells. When expressed in insect cells, poneratoxin was observed attached to cell membranes. Both synthetic and recombinant ponerotoxins were soluble below pH 4.5. The structure of synthetic poneratoxin was characterized by circular dichroism and solved by nuclear magnetic resonance. In an environment imitating a lipid bilayer, at pH within the range of insect hemolymph, synthetic poneratoxin has a V shape, with two a-helices connected by a b-turn. Insect larvae were paralyzed by injection of either of the purified toxins, with the recombinant one acting faster. The recombinant toxin-producing baculovirus reduced the average survival time of the insect host by 25 h compared with unmodified virus. Mass spectrometry analysis showed that the recombinant toxin has an N-terminal 21-residue extension, possibly improving its stability and/or stabilizing the membrane-bound state. The potential use of poneratoxin for the construction of biological insecticide is discussed.

Transcriptome Analysis in Venom Gland of the Predatory Giant Ant Dinoponera quadriceps: Insights into the Polypeptide Toxin Arsenal of Hymenopterans

“…Therefore, knowledge of venom composition is desirable not only to comprehend the pathophysiological basis of the effects and symptoms elicited by the sting of D. quadriceps but also to characterize proteins and peptides that will potentially be useful in the diagnosis and prospective treatment of chronic human diseases [5], [36]. The biotechnological applications of ant venom include not only the development of pharmaceuticals but also the development of bio-insecticides; the latter benefit is exemplified by poneratoxin, a small 25-residue neuropeptide that is active at the Na+- and K + -channels of invertebrates and was originally isolated in the venom of Paroponera clavata [29].…”

“…These symptoms are mostly attributable to a cocktail of low molecular weight organic compounds and pharmacologically active polypeptides that is present in the venom. Indeed, regarding the polypeptide core of the venom, short peptides isolated from Paraponera clavata venom (i.e., poneratoxins (PoTx)) have been proven to prolong the action of sodium channels and interfere with neurotransmission [28], [29]. To identify individual venom peptides and proteins in the venom of Dinoponera australis , Johnson and colleagues [30] conducted a proteomic survey via high performance liquid chromatography coupled with mass-spectrometry (HPLC/DAD/MS).…”

Expression, Delivery and Function of Insecticidal Proteins Expressed by Recombinant Baculoviruses

“…Numerous other venom neurotoxins have been expressed in baculoviruses. Most of these neurotoxins have been from scorpion venoms [ 42 , 43 , 49 , 60 , 63 , 65 , 66 ], but neurotoxins from the venoms of spiders [ 22 , 30 , 45 , 46 ], a hornet [ 41 ], an ant [ 59 ], and a sea anemone [ 30 , 45 , 46 ] have also been inserted into baculoviruses. In most cases, paralysis of infected host larvae and a reduction in survival time is achieved with expression of these neurotoxins.…”

“…Since many insecticidal proteins are usually secreted, they bear a signal peptide at the N-terminus that directs the protein into the endoplasmic reticulum, which is the first step in the pathway leading to secretion from the cell. For some of the neurotoxins selected for expression in baculoviruses, the only sequence data available was of the amino acid sequence of the mature peptide (e.g., [ 59 , 63 ]). Such a sequence would be missing the signal peptide, which is cleaved off upon translocation of the nascent toxin peptide chain into the endoplasmic reticulum.…”

“…Heterologous signal peptides from multiple sources have been used in baculoviruses expressing insecticidal proteins, including signal peptides from human beta-interferon [ 24 ], human placental alkaline phosphatase [ 78 ], D. melanogaster cuticle protein II [ 40 ], B. mori bombyxin [ 25 ], H. virescens juvenile hormone esterase [ 27 ], T. ni acidic juvenile hormone-suppressible hemolymph protein (AJSP-1; [ 22 ]), flesh fly sarcotoxin IA [ 40 , 69 ], honeybee melittin [ 45 ], M. sexta adipokinetic hormone [ 33 ], Lucilia cuprina (Australian sheep blowfly) chymotrypsin [ 33 ], AcMNPV GP67 [ 33 , 35 , 59 ], AcMNPV EGT [ 60 ], Spodoptera litura NPV EFP (envelope fusion protein) [ 66 ], and the venom neurotoxin signal peptides from the mite toxin encoded by tox21A [ 40 ] and neurotoxins AaIT, LqhIT2, and BjIT (from the scorpion Hottentota judaicus ) [ 33 ]. In two studies, signal peptides from different sources were compared to identify a sequence that drove optimal levels of protein secretion.…”