title: Sphingomyelinase D activity in model membranes: structural effects of in situ generation of ceramide-1-phosphate.
abstract: The toxicity of Loxosceles spider venom has been attributed to a rare enzyme, sphingomyelinase D, which transforms sphingomyelin to ceramide-1-phosphate. The bases of its inflammatory and dermonecrotic activity, however, remain unclear. In this work the effects of ceramide-1-phosphate on model membranes were studied both by in situ generation of this lipid using a recombinant sphingomyelinase D from the spider Loxosceles laeta and by pre-mixing it with sphingomyelin and cholesterol. The systems of choice were large unilamellar vesicles for bulk studies (enzyme kinetics, fluorescence spectroscopy and dynamic light scattering) and giant unilamellar vesicles for fluorescence microscopy examination using a variety of fluorescent probes. The influence of membrane lateral structure on the kinetics of enzyme activity and the consequences of enzyme activity on the structure of target membranes containing sphingomyelin were examined. The findings indicate that: 1) ceramide-1-phosphate (particularly lauroyl ceramide-1-phosphate) can be incorporated into sphingomyelin bilayers in a concentration-dependent manner and generates coexistence of liquid disordered/solid ordered domains, 2) the activity of sphingomyelinase D is clearly influenced by the supramolecular organization of its substrate in membranes and, 3) in situ ceramide-1-phosphate generation by enzymatic activity profoundly alters the lateral structure and morphology of the target membranes.
authors.affiliation: Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, Mexico.
authors.forename: Roberto P
authors.initials: RP
authors.lastname: Stock
authors.forename: Jonathan
authors.initials: J
authors.lastname: Brewer
authors.forename: Kerstin
authors.initials: K
authors.lastname: Wagner
authors.forename: Blanca
authors.initials: B
authors.lastname: Ramos-Cerrillo
authors.forename: Lars
authors.initials: L
authors.lastname: Duelund
authors.forename: Kit Drescher
authors.initials: KD
authors.lastname: Jernshøj
authors.forename: Lars Folke
authors.initials: LF
authors.lastname: Olsen
authors.forename: Luis A
authors.initials: LA
authors.lastname: Bagatolli
mesh_terms: D015081:2-Naphthylamine / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D002152:Calorimetry, Differential Scanning; D002518:Ceramides / Q000737:chemistry* / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D007848:Laurates / Q000378:metabolism; D008027:Light; D008567:Membranes, Artificial; D008856:Microscopy, Fluorescence; D010713:Phosphatidylcholines; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D012542:Scattering, Radiation; D013109:Sphingomyelins / Q000378:metabolism; D013112:Spiders / Q000201:enzymology; D013696:Temperature; D053835:Unilamellar Liposomes / Q000378:metabolism
chemical_list: D002518:Ceramides; D007848:Laurates; D008567:Membranes, Artificial; D010713:Phosphatidylcholines; D013109:Sphingomyelins; D053835:Unilamellar Liposomes; C065576:ceramide 1-phosphate; D002784:Cholesterol; D015081:2-Naphthylamine; D010727:Phosphoric Diester Hydrolases; C055160:sphingomyelin phosphodiesterase D; C017251:1,2-oleoylphosphatidylcholine; C065580:laurdan