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.

Investigation of the biophysical properties of a fluorescently modified ceramide-1-phosphate

“…POPC and C1P MLVs displayed a GP value between 0 and −0.1. These data are consistent with previously published data showing a GP value of ~ −0.5 for 5 mol% C1P (Stock et al, 2012). However, MLVs containing TopFluor-C1P showed significantly increased GP values: 0.2, 0.58, and 0.65, for 1, 3, and 5 mol% respectively, indicating that TopFluor-C1P creates a more rigid and ordered membrane structure.…”

Phospholipase D Toxins of Brown Spider Venom Convert Lysophosphatidylcholine and Sphingomyelin to Cyclic Phosphates

“…For example, CPA is a known signaling molecule with distinct agonistic effects from LPA on mammalian cells [39], and is an inhibitor of peroxisome proliferator-activated receptor gamma, a nuclear receptor that helps to regulate cell proliferation, apoptosis, and inflammation [40]. Also, conversion of SM to CC(1,3)P may disturb bilayer integrity and morphology [41], which could affect membrane asymmetry. Loss of membrane asymmetry has been proposed as a mechanism to induce the complement pathway of the innate immune response, which can result in cell lysis [4], [6], [42].…”

Ceramide 1-Phosphate Mediates Endothelial Cell Invasion via the Annexin a2-p11 Heterotetrameric Protein Complex

“…Further supporting this possibility, the affinity of annexin a2 for C-1-P was 14-fold greater than that of PA. One explanation for this difference could be the physical state of the membrane. C-1-P, for example, has been shown to induce domain formation in vesicles (56). C-1-P and PA also have different biophysical properties in membranes that may explain the difference in selectivity.…”