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The Hemiscorpius lepturus scorpion and brown spider Loxosceles intermedia represent a public health problem in Asia and America, respectively. Although distinct, these organisms contain similar toxins responsible for the principal clinical signs of envenomation. To better understand the properties of these toxins, we designed a study to compare recombinant Heminecrolysin (rHNC) and rLiD1, the major phospholipase D toxins of scorpion and spider venom, respectively. Using a competitive ELISA and a hemolytic inhibition test, we come to spot a cross reaction between scorpion and spider venoms along with an epitopic similarity between rHNC and rLiD1 associated with neutralizing antibodies. Results show that the ability of the rHNC to hydrolyze lysophosphatidylcholine (LPC) is equivalent to that of rLiD1 to hydrolyze sphingomyelin and vice-versa. rHNC exclusively catalyze transphosphatidylation of LPC producing cyclic phosphatidic acid (cPA). The in-silico analysis of hydrogen bonds between LPC and toxins provides a possible explanation for the higher transphosphatidylase activity of rHNC. Interestingly, for the first time, we reveal that lysophosphatidic acid (LPA) can be a substrate for both enzymes using cellular and enzymatic assays. The finding of the usage of LPA as a substrate as well as the formation of cPA as an end product could shed more light on the molecular basis of Hemiscorpius lepturus envenomation as well as on loxoscelism.

International audience; In a previous study, we reported the identification of Hemilipin, the first secreted heterodimeric phospholipase A2 (sPLA2) from Hemiscorpius lepturus scorpion venom and demonstrated its effective inhibition of all angiogenesis key steps in vitro and in vivo. Here, we aimed to characterize a second sPLA2, Hemilipin2, from the same venom and to elucidate its antiangiogenic effect. The protein was purified by chromatography separation and analyzed by MALDI/TOF mass spectrometry. Its N terminal amino acid sequence was determined by Edman degradation method and the enzymatic activity by fatty acids release assay. Hemilipin2 antiangiogenic activity was investigated by studying its effect in vitro on adhesion, migration and capillary like tube formation of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Pulmonary Artery Endothelial Cells (HPAECs); and in vivo on the chick embryo chorioallantoic membrane (CAM) assay. Data to be presented show that Hemilipin2 is heterodimeric composed by two subunits: the large one has a molecular weight of 12,866 and the small one of 2461 a.m.u. It has a strong calcium-dependent PLA2 activity and impacts angiogenesis in vitro and in vivo without showing any cytotoxic or apoptotic signs. Its chemical modification with p-Bromophenacyl Bromide abolishes the enzymatic activity without affecting the antiangiogenic effect. Furthermore, it has been proved that Hemilipin2 small subunit was able to inhibit blood vessel formation both in vitro and in vivo. These findings may serve as a starting point for the designing of a new generation of specific inhibitor of human angiogenesis at different steps. (C) 2016 Elsevier Ltd. All rights reserved.

International audience; Phospholipases A(2) (PLA(2)) are enzymes which specifically hydrolyze the sn-2 acyl ester bond of phospholipids producing free fatty acids and lysophospholipids. The secreted PLA(2) (sPLA(2)) are the most common types of PLA(2) purified from the snake venom, mammalian pancreatic juice and other sources. They display a variety of toxic actions and biological activities, including antitumoral and antiangiogenic effects. In this study, we report the isolation, characterization and the antiangiogenic activity of Hemilipin, a novel sPLA(2) extracted from Hemiscomius lepturus venom, the most dangerous scorpion in Iran. Hemilipin was purified by HPLC and analyzed by MALDI TOF/MS. The primary structure was determined by EDMAN degradation method and the PLA(2) activity by titration of fatty acids released from the egg yolk phospholipids. Its antiangiogenic activity was studied in vitro by evaluating effects on apoptosis, Matrigel angiogenesis, migration and adhesion of human umbilical vein endothelial cells (HUVECs) and human pulmonary artery endothelial cells (HPAECs) and in vivo by the chorioallantoic membrane (CAM) assay. Mass spectrometry profile showed that Hemilipin is heterodimeric and the PLA(2) test demonstrated its strong hydrolytic activity. N-terminal aminoacid sequence highlighted a significant homology of Hemilipin's small and large subunits with other sPLA(2) group III. Hemilipin had no effect on apoptosis, but strongly impacted angiogenesis both in vitro and in vivo. Our results demonstrate that this novel non toxic sPLA(2) could be a new tool to disrupt at different steps human angiogenesis. (C) 2015 Elsevier Ltd. All rights reserved.

International audience; We have previously identified Heminecrolysin, a sphingomyelinase D (SMaseD), as the major protein responsible for the main pathological effects observed following Hemiscorpius (H.) lepturus scorpion envenomation. We aimed herein to further investigate the kinetics and molecular mechanisms triggered by Heminecrolysin to initiate hematological disorders and inflammatory reaction. We show that Heminecrolysin highly hydrolyzes lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA) and choline, with a Vmax = 1481 ± 51 μmol/min/mg and a Km = 97 ± 16.78 μM, at a much lesser extend sphingomyelin but not phosphatidylcholine substrates. Its lysophospholipase D (lysoPLD) catalytic efficiency, up to three orders of magnitude higher, comparatively to spider's SMaseDs (newly referred as phospholipases D; PLDs), could explain its strong hemolytic capacity. Chelating agents such as EDTA, EGTA, and 1, 10-phenantroline blocked Heminecrolysin-induced LPC hydrolysis at 98, 48, and 70% respectively. Hemolysis blockade occurs only when the toxin is added to erythrocytes in the presence of serum, source of LPC and complement, indicating that the production of LPA and the presence of complement are mandatory for hemolysis. Moreover, we show that Heminecrolysin efficiently binds to erythrocyte's membrane and provokes phosphatidylserine (PS) translocation without cleavage of glycophorin A, suggesting that, unlike spider's PLDs, complement was activated only via the classical pathway. Interestingly, Heminecrolysin was found to induce PS exposure on human nucleated Jurkat T cells, to stimulate secretion of the pro-inflammatory (TNF-α, IL-6), and anti-inflammatory (IL-10) cytokines by human monocytes, and to provoke a disseminated intravascular coagulation on chick embryo chorioallantoic membrane model system. Taken together, our results indicate that Heminecrolysin evokes the major characteristic clinical features of H. lepturus envenomation by using mainly its lysoPLD, rather than its SMaseD's, activity.

Hemitoxin (HTX) is a new K+ channel blocker isolated from the venom of the Iranian scorpion Hemiscorpius lepturus. It represents only 0.1% of the venom proteins, and displaces [125 I]alpha-dendrotoxin from its site on rat brain synaptosomes with an IC50 value of 16 nm. The amino acid sequence of HTX shows that it is a 35-mer basic peptide with eight cysteine residues, sharing 29-69% sequence identity with other K+ channel toxins, especially with those of the alphaKTX6 family. A homology-based molecular model generated for HTX shows the characteristic alpha/beta-scaffold of scorpion toxins. The pairing of its disulfide bridges, deduced from MS of trypsin-digested peptide, is similar to that of classical four disulfide bridged scorpion toxins (Cys1-Cys5, Cys2-Cys6, Cys3-Cys7 and Cys4-Cys8). Although it shows the highest sequence similarity with maurotoxin, HTX displays different affinities for Kv1 channel subtypes. It blocks rat Kv1.1, Kv1.2 and Kv1.3 channels expressed in Xenopus oocytes with IC50 values of 13, 16 and 2 nM, respectively. As previous studies have shown the critical role played by the beta-sheet in Kv1.3 blockers, we suggest that Arg231 is also important for Kv1.3 versus Kv1.2 HTX positive discrimination. This article gives information on the structure-function relationships of Kv1.2 and Kv1.3 inhibitors targeting developing peptidic inhibitors for the rational design of new toxins targeting given K+ channels with high selectivity.

International audience

International audience; We have constructed a cDNA library from venom glands of the scorpion Buthus occitanus tunetanus and cloned a DNA sequence that encodes an alpha-toxin. This clone was efficiently expressed in Escherichia coli as a fusion protein with two Ig-binding (Z) domains of protein A from Staphylococcus aureus. After CNBr treatment of the fusion protein and HPLC purification, we obtained approximately 1 mg recombinant apha-toxin/l bacterial culture. The toxin, called Bot XIV, displays no toxicity towards mammals but is active towards insects as shown by its paralytic activity against Blatella germanica cockroach and by electrophysiological studies on Periplaneta americana cockroaches. The Bot XIV protein fused to two Z domains is highly immunogenic in mice and induces production of antisera that specifically recognize and neutralize highly toxic components that had been injected into mice. This fusion protein could be very useful for development of potent protective antisera against scorpion venoms.

The effects of insect toxin Bot IT2, purified from the venom of the scorpion Buthus occitanus tunetanus, were investigated on the isolated giant axon and on isolated dorsal unpaired median (DUM) neurone of the cockroach Periplaneta americana under current- and voltage-clamp conditions, using the double-oil-gap technique and the patch-clamp technique, respectively. In both preparations, Bot IT2, induces a limited depolarization together with the development of a repetitive activity in axon and an increase of spontaneous discharge frequency in DUM neurone. After artificial hyperpolarization to the normal resting level, plateau potentials can be evoked in both preparations. Under voltage-clamp conditions, Bot IT2 induces a similar effect in axon and DUM neurone by acting specifically on sodium channels. The peak sodium current is decreased and simultaneously a new current with very slow activation-deactivation kinetics is developed. Voltage dependence of this slow current is not very different from that of the control. The inactivation of the fast component is incomplete because it is masked by the development of the slow component. These results suggest that Bot IT2 modifies the kinetics of insect sodium channel activation, and the transformation of normal fast channels into slow ones is discussed.

Serotherapy against Hemiscorpius (H.) lepturus scorpion sting is based on the administration of equine polyvalent antivenom prepared against a mixture of six venoms. In a previous study, we reported the identification of Heminecrolysin, a 33 kDa H. lepturus venom protein endowed with a sphingomyelinase D, hemolytic and dermonecrotic activities. We aimed herein to investigate the capacity of Heminecrolysin to generate antibodies able to neutralize the major physiopathological properties of H. lepturus envenomation, e.g. hemolysis and dermonecrosis. The efficiency of anti-Heminecrolysin antibodies was compared to that of anti-whole venom. Our results demonstrated that Heminecrolysin elicits high levels of specific IgGs. Anti-Heminecrolysin, similarly to anti-whole venom antibodies, totally inhibited H. lepturus hemolytic effect when up to 5 times the half maximal effective concentration of venom were used. Phosphatidylserine exposure on the external lipid monolayer of human red blood cells treated with whole venom was also fully blocked by both anti-sera. Experimental envenomation of rabbits showed that anti-Heminecrolysin antibodies were as potent as anti-H. lepturus antibodies to neutralize dermonecrotic effects when up to 4 times the minimal necrotic dose of venom were injected. However, inflammatory reaction was better controlled with anti-whole venom sera. In conclusion, Heminecrolysin elicits protective antibodies of comparable potency to those elicited by immunization with whole venom.

International audience; BACKGROUND: Recurrent bacterial and fungal infections, eczema, and increased serum IgE levels characterize patients with the hyper-IgE syndrome (HIES). Known genetic causes for HIES are mutations in signal transducer and activator of transcription 3 (STAT3) and dedicator of cytokinesis 8 (DOCK8), which are involved in signal transduction pathways. However, glycosylation defects have not been described in patients with HIES. One crucial enzyme in the glycosylation pathway is phosphoglucomutase 3 (PGM3), which catalyzes a key step in the synthesis of uridine diphosphate N-acetylglucosamine, which is required for the biosynthesis of N-glycans. OBJECTIVE: We sought to elucidate the genetic cause in patients with HIES who do not carry mutations in STAT3 or DOCK8. METHODS: After establishing a linkage interval by means of SNPchip genotyping and homozygosity mapping in 2 families with HIES from Tunisia, mutational analysis was performed with selector-based, high-throughput sequencing. Protein expression was analyzed by means of Western blotting, and glycosylation was profiled by using mass spectrometry. RESULTS: Mutational analysis of candidate genes in an 11.9-Mb linkage region on chromosome 6 shared by 2 multiplex families identified 2 homozygous mutations in PGM3 that segregated with disease status and followed recessive inheritance. The mutations predict amino acid changes in PGM3 (p.Glu340del and p.Leu83Ser). A third homozygous mutation (p.Asp502Tyr) and the p.Leu83Ser variant were identified in 2 other affected families, respectively. These hypomorphic mutations have an effect on the biosynthetic reactions involving uridine diphosphate N-acetylglucosamine. Glycomic analysis revealed an aberrant glycosylation pattern in leukocytes demonstrated by a reduced level of tri-antennary and tetra-antennary N-glycans. T-cell proliferation and differentiation were impaired in patients. Most patients had developmental delay, and many had psychomotor retardation. CONCLUSION: Impairment of PGM3 function leads to a novel primary (inborn) error of development and immunity because biallelic hypomorphic mutations are associated with impaired glycosylation and a hyper-IgE-like phenotype.