All experiments involving animals were performed in accordance with the ethical committee on research animal care agreement in Pasteur Institute of Iran (approval number: IR

All experiments involving animals were performed in accordance with the ethical committee on research animal care agreement in Pasteur Institute of Iran (approval number: IR.PII.REC.1394.86). beta/alpha-barrel. The lethal dose 50 (LD50) and dermonecrotic activities of Hl-RecPLD1 were decided as 3.1 g/mouse and 0.7 cm2at 1 g respectively. It is the first Rabbit Polyclonal to OR2L5 report indicating that a comparable molecular evolutionary mechanism has occurred in both American brown spiders and this Iranian scorpion. In conclusion, Hl-RecPLD1 is usually a highly active phospholipase D, which would be considered as the lethal dermonecrotic toxin inH. lepturusvenom. Keywords:Hemiscorpius lepturus, scorpion, phospholipase D, homology, brown spiders, Loxoscelidae family, lethality, dermonecrotic activity == 1. Introduction == Scorpionism is one of the most important public health problems, including 2.3 billion inhabitants in tropical, subtropical and temperate regions [1]. Scorpion stings have been frequently reported in Iran [2] with the most common incidence occurring in the Khuzestan province (541/100,000 people; 2.2% Lanraplenib of the population) [3,4]. Among all scorpion species in Iran, there are six species that are medically important and responsible for scorpionism, includingH. lepturus,Odontobuthus doriae, Mesobuthus eupeus,Androctonus crassicauda,Buthotus saulcyiandButhotus sach[5]. The most medically important scorpion in Iran isH. lepturus,which belongs to Hemiscorpiidae family [6]. ExcludingH. lepturus, almost all of the other venomous scorpion species in Iran belong to the large family of Buthidae [7]. Although only 15% of scorpion stings have been attributed toH. lepturus,it is the most lethal scorpion and accounted for 89% of deaths [8]. The mortality rate fromH. lepturusenvenomation is usually approximately 60 occasions higher than the other Iranian scorpions [8]. The most severe toxicity has been primarily reported in children [9]. Unlike other scorpions, the venom ofH. lepturusis highly cytotoxic which could be the reason for its complicated clinical manifestations in envenomed people [10]. The sting ofH. lepturusleads to numerous symptoms, including cutaneous Lanraplenib reactions varying from undetectable small areas of erythema to large areas of dermonecrotic lesions at the sting site. In severe cases, the scorpion sting can trigger systemic effects characterized by persistent inflammation, vascular leakage, platelet aggregation, cardiovascular disease, severe hemolysis and hematuria, which can lead to renal failure [8,11,12,13]. These systemic disturbances may be attributable to some enzymatic components in the venom ofH. lepturus[12]. Moreover, an increased level of liver enzymes (i.e., aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP)) has also been reported as a potential result of this venom, indicating hepatocellular damage [10]. Our comprehensive literature review revealed that the major clinical indicators ofH. lepturusenvenomation [8,11,12,13,14] are similar to those caused byLoxoscelesgenera (brown spiders) [15,16,17]. This observation may indicate the existence of major homologous toxins in both venoms. The dermonecrotic toxins are major molecules in the crude venom of brown spiders, being responsible for many clinical complications and deaths in humans. These dermonecrotic toxins were characterized as sphingomyelinase D (SMase D) in previous studies [18,19,20,21]. The toxins are able to hydrolyze sphingomyelin (SM), the major component of the outer leaflet of plasma membranes, into choline and ceramide 1-phosphate [18,19,20,21]. Sphingomyelinase D in brown spiders can act as a phospholipase D and its metabolites are important lipid mediators involved in several pathological and physiological processes [22,23,24,25,26]. It has been shown that different SMase D isoforms are simultaneously existed in the venom of a brown spider. These isoforms have various levels of sphingomyelinase, hemolytic, and lethal activities [19,21,26]. A similar SMase D molecule, designated as Heminecrolysin (HNc), has been partially sequenced and characterized as a dermonecrotic toxin inH. lepturusvenom. However, its lethal activity has not been assessed yet [27]. According to our best of knowledge, no studies have been conducted to characterization ofH. lepturusregarding the reason of venom lethality. Thus, it is necessitated to discover a responsible lethal toxin Lanraplenib inH. lepturusvenom. In the present study, we identified a novel recombinant phospholipase D responsible for the lethality and dermonecrotic activity ofH. lepturusvenom using both bioinformatic and functional assays. == 2. Results == == 2.1. Selection and Characterization of the Candidate Open Reading Frame (ORF) and Main Chain == Based on the results of homology analysis using protein-protein basic local alignment search tool (BLASTP), the best ORF with highest identity was selected. The candidate ORF and its main chain were determined as chains with lengths of 324 and 286 amino acids respectively (Figure 1). Hl-PLD1 demonstrated the highest sequence similarity with the isoforms of PLD fromL. intermedia(identity 46%48%). Other PLDs.