title: Mechanism of induction of complement susceptibility of erythrocytes by spider and bacterial sphingomyelinases.
abstract: We have recently shown that the sphingomyelinase toxins P1 and P2 from the venom of the spider Loxosceles intermedia induce complement (C)-dependent lysis of autologous erythrocytes by induction of the cleavage of cell surface glycophorins through activation of an endogenous metalloproteinase facilitating the activation of the alternative pathway of C. Phospholipase D (PLD) from Corynebacterium pseudotuberculosis shows some degree of homology with the spider sphingomyelinases and can induce similar clinical symptoms to those observed after spider envenomation. The aim of this study was to investigate if the bacterial PLD-induced haemolysis of human erythrocytes was C dependent and if cleavage of glycophorins occurred. We show here that haemolysis of both PLD- and P1-treated human erythrocytes was C dependent, but while PLD-mediated haemolysis was dependent on activation of the classical pathway of C, P1 induced lysis via both the classical and alternative pathways. P1, but not PLD, induced cleavage of glycophorins and no change in expression of complement regulators was induced by either of the toxins. In both cases, annexin V binding sites were exposed, suggesting that the membrane asymmetry had been disturbed causing exposure of phosphatidylserine to the cell surface. Our results suggest that C susceptibility induced by L. intermedia and C. pseudotuberculosis PLD is a result of exposure of phosphatidylserine, and the higher potency of P1 toxin can be explained by its additional effect of cleavage of glycophorins.
authors.affiliation: Laboratório de Imunoquímica, Instituto Butantan, São Paulo, Brazil. dvtambourgi@yahoo.com
authors.forename: Denise V
authors.initials: DV
authors.lastname: Tambourgi
authors.forename: Marcelo
authors.initials: M
authors.lastname: De Sousa Da Silva
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Billington
authors.forename: Rute M
authors.initials: RM
authors.lastname: Gonçalves De Andrade
authors.forename: Fábio C
authors.initials: FC
authors.lastname: Magnoli
authors.forename: J Glenn
authors.initials: JG
authors.lastname: Songer
authors.forename: Carmen W
authors.initials: CW
authors.lastname: Van Den Berg
mesh_terms: D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D001665:Binding Sites; D003167:Complement Activation / Q000187:drug effects*; D016925:Corynebacterium pseudotuberculosis; D004306:Dose-Response Relationship, Immunologic; D004910:Erythrocyte Membrane / Q000378:metabolism; D004912:Erythrocytes / Q000187:drug effects / Q000276:immunology; D006021:Glycophorins / Q000378:metabolism; D006461:Hemolysis / Q000187:drug effects*; D006801:Humans; D010739:Phospholipase D / Q000494:pharmacology*; D013108:Sphingomyelin Phosphodiesterase / Q000494:pharmacology*; D013111:Spider Venoms / Q000494:pharmacology*; D013112:Spiders
chemical_list: D017304:Annexin A5; D006021:Glycophorins; D013111:Spider Venoms; D013108:Sphingomyelin Phosphodiesterase; D010739:Phospholipase D