title: The pathological effects of Heminecrolysin, a dermonecrotic toxin from Hemiscorpius lepturus scorpion venom are mediated through its lysophospholipase D activity.
abstract: 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.
authors.affiliation: Laboratoire Venins et Biomolécules Thérapeutiques LR11IPT08, Institut Pasteur de Tunis, Université de Tunis El Manar, Tunis 1002, Tunisia. lamia.borchani@pasteur.rns.tn
authors.forename: Lamia
authors.initials: L
authors.lastname: Borchani
authors.forename: Atfa
authors.initials: A
authors.lastname: Sassi
authors.forename: Haifa
authors.initials: H
authors.lastname: Ben Gharsa
authors.forename: Ines
authors.initials: I
authors.lastname: Safra
authors.forename: Delavar
authors.initials: D
authors.lastname: Shahbazzadeh
authors.forename: Zakaria
authors.initials: Z
authors.lastname: Ben Lasfar
authors.forename: Mohamed
authors.initials: M
authors.lastname: El Ayeb
mesh_terms: D000818:Animals; D002642:Chick Embryo; D002794:Choline / Q000378:metabolism; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism; D006021:Glycophorins / Q000378:metabolism; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D016753:Interleukin-10 / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D019169:Jurkat Cells; D008244:Lysophosphatidylcholines / Q000037:antagonists & inhibitors / Q000378:metabolism; D008246:Lysophospholipids / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D012604:Scorpion Venoms / Q000633:toxicity*; D012605:Scorpions; D013112:Spiders; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D000099189:Lysophospholipase D
chemical_list: D002794:Choline; D006021:Glycophorins; D016753:Interleukin-10; D015850:Interleukin-6; D008244:Lysophosphatidylcholines; D008246:Lysophospholipids; D010718:Phosphatidylserines; D010727:Phosphoric Diester Hydrolases; D012604:Scorpion Venoms; D014409:Tumor Necrosis Factor-alpha; D000099189:Lysophospholipase D; C055160:sphingomyelin phosphodiesterase D; C032881:lysophosphatidic acid