Certaines toxines présentes dans les venins de scorpions sont beaucoup plus toxiques pour les ins... more Certaines toxines présentes dans les venins de scorpions sont beaucoup plus toxiques pour les insectes que pour les autres classes animales et présentent une affinité élevée pour les canaux Na+ dépendants du potentiel membranaire. Plusieurs toxines anti-insectes ont été purifiées du venin de Buthus occitanus tunetanus. En se basant sur les séquences en acides aminés et en utilisant l'amplification par la technique RACE PCR (Rapid amplification of cDNA ends), on a pu cloner, identifier et séquencer l'ADNc d'une toxine anti-insecte purifiée du venin de Buthus occitanus tunetanus. La séquence nucléotidique déduite code pour une toxine de 60 acides aminés, identique à la toxine anti-insecte BotIT2 1. BotIT2 est similaire aux toxines anti-insectes contracturantes dans son mode d'action, elle est plutôt homologue aux toxines flasques dans sa structure primaire 1 .
We have previously identified Heminecrolysin, a sphingomyelinase D (SMaseD), as the major protein... more 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.
Toxicon : official journal of the International Society on Toxinology, 1997
One contractive and two depressant toxins active on insect were purified by high-performance liqu... more One contractive and two depressant toxins active on insect were purified by high-performance liquid chromatography from the venom of Buthus occitanus tunetanus (Bot). The two depressant toxins, BotIT4 and BotIT5, differ only at position 6 (Arg for Lys) and are equally toxic to insects (LD50 to Blatella germanica = 110 ng/100 mg body weight). They show a strong antigenic cross-reaction with a depressive toxin from Leiurus quinquestriatus quinquestriatus (LqqIT2). The two toxins are able to inhibit with high affinity (K0.5 between 2 and 3 nM) the specific binding of the radioiodinated excitatory insect toxin (125I-AaHIT) on its receptor site on Periplaneta americana synaptosomal membranes. These toxins depolarize the cockroach axon, irreversibly block the action potential, and slow down and very progressively block the transmembrane transient Na+ current. The contracturant toxin BotIT1 is highly toxic to B. germanica (LD50 = 60 ng/ 100 mg body weight) and barely toxic to mice (LD50 = ...
Serotherapy against Hemiscorpius (H.) lepturus scorpion sting is based on the administration of e... more 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.
Recurrent bacterial and fungal infections, eczema, and increased serum IgE levels characterize pa... more 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. We sought to elucidate the genetic cause in patients with HIES who do not carry mutations in STAT3 or DOCK8. 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. 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. 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.
Numerous toxins from scorpion venoms are much more toxic to insects than to other animal classes,... more Numerous toxins from scorpion venoms are much more toxic to insects than to other animal classes, and possess high affinity to Na+ channels. Many of them active on insects were purified from the venom of Buthus occitanus tunetanus. Using amino acid sequences of BotIT2 and RACE-PCR amplification (Rapid amplification of cDNA ends) technique, we isolated, identified and sequenced the nucleotide sequence from the venom glands of the scorpion Buthus occitanus tunetanus. The cDNA encodes a precursor of an insect toxin of 60 amino acid residues. The deduced nucleotide sequence toxin was identical to the determined amino acid sequence of BotIT2. BotIT2 is more similar to the excitatory toxins in its mode of action and to the depressant toxins in its primary structure.
Certaines toxines présentes dans les venins de scorpions sont beaucoup plus toxiques pour les ins... more Certaines toxines présentes dans les venins de scorpions sont beaucoup plus toxiques pour les insectes que pour les autres classes animales et présentent une affinité élevée pour les canaux Na+ dépendants du potentiel membranaire. Plusieurs toxines anti-insectes ont été purifiées du venin de Buthus occitanus tunetanus. En se basant sur les séquences en acides aminés et en utilisant l'amplification par la technique RACE PCR (Rapid amplification of cDNA ends), on a pu cloner, identifier et séquencer l'ADNc d'une toxine anti-insecte purifiée du venin de Buthus occitanus tunetanus. La séquence nucléotidique déduite code pour une toxine de 60 acides aminés, identique à la toxine anti-insecte BotIT2 1. BotIT2 est similaire aux toxines anti-insectes contracturantes dans son mode d'action, elle est plutôt homologue aux toxines flasques dans sa structure primaire 1 .
We have previously identified Heminecrolysin, a sphingomyelinase D (SMaseD), as the major protein... more 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.
Toxicon : official journal of the International Society on Toxinology, 1997
One contractive and two depressant toxins active on insect were purified by high-performance liqu... more One contractive and two depressant toxins active on insect were purified by high-performance liquid chromatography from the venom of Buthus occitanus tunetanus (Bot). The two depressant toxins, BotIT4 and BotIT5, differ only at position 6 (Arg for Lys) and are equally toxic to insects (LD50 to Blatella germanica = 110 ng/100 mg body weight). They show a strong antigenic cross-reaction with a depressive toxin from Leiurus quinquestriatus quinquestriatus (LqqIT2). The two toxins are able to inhibit with high affinity (K0.5 between 2 and 3 nM) the specific binding of the radioiodinated excitatory insect toxin (125I-AaHIT) on its receptor site on Periplaneta americana synaptosomal membranes. These toxins depolarize the cockroach axon, irreversibly block the action potential, and slow down and very progressively block the transmembrane transient Na+ current. The contracturant toxin BotIT1 is highly toxic to B. germanica (LD50 = 60 ng/ 100 mg body weight) and barely toxic to mice (LD50 = ...
Serotherapy against Hemiscorpius (H.) lepturus scorpion sting is based on the administration of e... more 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.
Recurrent bacterial and fungal infections, eczema, and increased serum IgE levels characterize pa... more 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. We sought to elucidate the genetic cause in patients with HIES who do not carry mutations in STAT3 or DOCK8. 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. 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. 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.
Numerous toxins from scorpion venoms are much more toxic to insects than to other animal classes,... more Numerous toxins from scorpion venoms are much more toxic to insects than to other animal classes, and possess high affinity to Na+ channels. Many of them active on insects were purified from the venom of Buthus occitanus tunetanus. Using amino acid sequences of BotIT2 and RACE-PCR amplification (Rapid amplification of cDNA ends) technique, we isolated, identified and sequenced the nucleotide sequence from the venom glands of the scorpion Buthus occitanus tunetanus. The cDNA encodes a precursor of an insect toxin of 60 amino acid residues. The deduced nucleotide sequence toxin was identical to the determined amino acid sequence of BotIT2. BotIT2 is more similar to the excitatory toxins in its mode of action and to the depressant toxins in its primary structure.
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Papers by L. Borchani