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.
Highlights
Spiders of the genus Loxosceles are considered of medical importance because they are capable of causing severe local and occasionally systemic envenomation in humans [1,2,3]
Morphological changes [10] and increased susceptibility to complement-mediated hemolysis of red blood cells resulting from SMD action have been reported [11], demonstrating that SM is available for enzymatic action. van Meeteren et al have argued that the lysophospholipase activity of the enzyme on circulating lysophospholipids underlies the inflammatory and dermonecrotic effects of the venom [12] and find a role for the sphingomyelinase D activity unlikely [13]
Dragulev et al have shown that recombinant SMD by itself can elicit production of proinflammatory factors in the absence of any source of exogenous lysophospholipids [14]. The latter favor the idea that the sphingomyelinase D activity of the enzyme is important in its overall toxicity with the implication that ceramide-1-phosphate plays a key role in the cellular response that leads to the clinical manifestations
Summary
Spiders of the genus Loxosceles are considered of medical importance because they are capable of causing severe local and occasionally systemic envenomation in humans [1,2,3]. Dragulev et al have shown that recombinant SMD by itself can elicit production of proinflammatory factors in the absence of any source of exogenous lysophospholipids [14] The latter favor the idea that the sphingomyelinase D activity of the enzyme is important in its overall toxicity with the implication that ceramide-1-phosphate plays a key role in the cellular response that leads to the clinical manifestations. These proposals are not mutually exclusive [15] and SMD has been recently suggested as a useful new probe in the study of the effects of SM metabolism at the cellular level [16]
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