Sphingomyelin-rich domains are sites of lysenin oligomerization: Implications for raft studies

Abstract

Lysenin is a self-assembling, pore-forming toxin which specifically recognizes sphingomyelin. Mutation of tryptophan 20 abolishes lysenin oligomerization and cytolytic activity. We studied the interaction of lysenin WT and W20A with sphingomyelin in membranes of various lipid compositions which, according to atomic force microscopy studies, generated either homo- or heterogeneous sphingomyelin distribution. Liposomes composed of SM/DOPC, SM/DOPC/cholesterol and SM/DPPC/cholesterol could bind the highest amounts of GST-lysenin WT, as shown by surface plasmon resonance analysis. These lipid compositions enhanced the release of carboxyfluorescein from liposomes induced by lysenin WT, pointing to the importance of heterogeneous sphingomyelin distribution for lysenin WT binding and oligomerization. Lysenin W20A bound more weakly to sphingomyelin-containing liposomes than did lysenin WT. The same amounts of lysenin W20A bound to sphingomyelin mixed with either DOPC or DPPC, indicating that the binding was not affected by sphingomyelin distribution in the membranes. The mutant lysenin had a limited ability to penetrate hydrophobic region of the membrane as indicated by measurements of surface pressure changes. When applied to detect sphingomyelin on the cell surface, lysenin W20A formed large conglomerates on the membrane, different from small and regular clusters of lysenin WT. Only lysenin WT recognized sphingomyelin pool affected by formation of raft-based signaling platforms. During fractionation of Triton X-100 cell lysates, SDS-resistant oligomers of lysenin WT associated with membrane fragments insoluble in Triton X-100 while monomers of lysenin W20A partitioned to Triton X-100-soluble membrane fractions. Altogether, the data suggest that oligomerization of lysenin WT is a prerequisite for its docking in raft-related domains.

Lipid Raft Redox Signaling: Molecular Mechanisms in Health and Disease

“…As summarized in Table 3, these LR signaling platforms include phosphorylation or transphosphorylation signaling platforms (1,49,74,97,105,279,287,317,329,405,436,439), GPCR raft signaling platforms (61,89,159,179,280,290), TCR signaling platforms (2,193,216,232,291,311,363,393), Ca 2 + channel signaling platforms (10,108,111,334,403,407), PI(4,5)P(2) rafts (54,224), STIM1 raft clustering at ER-plasma membrane junctions (7,108,187,293), cadiolipin platforms on mitochondria (366), raft-cytoskeleton nanodomains and macrodomains (218,332), and LR redox signaling platforms (191,192,234,235,283,306,…”

LPS induces phosphorylation of actin‐regulatory proteins leading to actin reassembly and macrophage motility

“…Samples were mounted in mowiol/DABCO and examined under a Leica confocal microscope with sequential excitation of FITC and Texas Red or TRITC to avoid crossover of their fluorescence. Colocalization of TLR4 and CD14 was analyzed using Leica Application Suite AF software which calculated Pearson's correlation coefficient and overlap coefficient essentially as described earlier [Kulma et al, 2010]. At least 40 cells from two independent experiments were analyzed for each variant.…”

Crucial Role of Perfringolysin O D1 Domain in Orchestrating Structural Transitions Leading to Membrane-perforating Pores

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