Bidirectional Control of Sphingomyelinase Activity and Surface Topography in Lipid Monolayers

Abstract

Lipid lateral organization is increasingly found to modulate membrane-bound enzymes. We followed in real time the reaction course of sphingomyelin (SM) degradation by Bacillus cereus sphingomyelinase (SMase) of lipid monolayers by epifluorescence microscopy. There is evidence that formation of ceramide (Cer), a lipid second messenger, drives structural reorganization of membrane lipids. Our results provide visual evidence that SMase activity initially alters surface topography by inducing phase separation into condensed (Cer-enriched) and expanded (SM-enriched) domains. The Cer-enriched phase grows steadily as the reaction proceeds at a constant rate. The surface topography derived from the SMase-driven reaction was compared with, and found to differ from, that of premixed SM/Cer monolayers of the same lipid composition, indicating that substantial information content is stored depending on the manner in which the surface was generated. The long-range topographic changes feed back on the kinetics of Smase, and the onset of condensed-phase percolation is temporally correlated with a rapid drop of reaction rate. These observations reveal a bidirectional influence and communication between effects taking place at the local molecular level and the supramolecular organization. The results suggest a novel biocatalytic-topographic mechanism in which a surface enzymatic activity can influence the function of amphitropic proteins important for cell function.

Recent advances in the immunobiology of ceramide

“…The first direct visual evidence for sphingomyelinase-induced formation of ceramide-enriched domains in sphingomyelin monolayer under precise control of the surface intermolecular packing has been provided recently (Fanani et al, 2002). Furthermore, it has been indicated that lateral enzyme-specific out-of equilibrium organization of lipid domains represents a new level of signal transduction from local (nm) to long-range (μm) scales (Hartel et al, 2005).…”

The many faces (and phases) of ceramide and sphingomyelin II – binary mixtures

“…Cer-induced domain segregation in several lipid mixtures with glycerophospholipids (Huang et al 1996;Carrer and Maggio 1999;Veiga et al 1999;Fanani et al 2002;Hsueh et al 2002;Sot et al 2005Sot et al , 2006Carrer et al 2006) has been reported using different biophysical approaches [see Alonso (2006, 2009) for reviews]. Cers induce high-temperature melting domains in mixtures where phospholipids display low (palmitoyl-oleoyl-PC) (Hsueh et al 2002) and high (dipalmitoyl-PC or dielaidoylphosphatidylethanolamine) (Carrer and Maggio 1999;Sot et al 2005) gel-to-fluid phase transition temperatures.…”

“…A microscopy study combining DSC, fluorescence spectroscopy, and confocal fluorescence on the interaction of egg Cer with its relative egg SM showed the segregation of detergentresistant Cer-enriched domains (Sot et al 2006). In either premixed or enzyme-generated systems with bovine brain SM [mainly stearic (18:0) and lignoceric (24:0) acyl chains], Cer induces morphologically different Cer-enriched domains, depending on how the mixture was generated (Fanani et al 2002De Tullio et al 2008). Furthermore, different types of Cer-enriched domains are enzymatically formed in the presence of cholesterol, showing high solubility in cholesterol-rich membranes (Silva et al 2009;Ale et al 2012).…”

“…For example, at the local nm level, molecular immiscibility (or ideal full miscibility) in binary monolayers (with or without macroscopic phase domain separation on the μm scale range) can be inferred by the existence of smooth surface pressure-area isotherms, linear additive variation of mean molecular area, and dipole potential density as a function of the film composition, together with composition-invariant collapse pressures (Phillips 1972;Brockman 1994;Maggio et al 1997;Carrer and Maggio 2001;Maggio 2004). In binary or ternary systems, lateral immiscibility can be visually observed on the μm scale range by the presence of segregated phase or compositional domains (McConnell 1990;Möhwald 1995;Maggio 2000, 2002;Fanani et al 2002;Rosetti et al 2003). However, when the system is rather complex regarding composition or surface topography, the emergence of lateral thermodynamic tensions and interfacial energy terms blurs away and impairs the detection of molecular cooperativity at the local level.…”

“…richly featured surface topography on the mesoscopic level, with coexistence of immiscible domains of different composition and phase state Maggio 2000, 2002;Fanani et al 2002;Rosetti et al 2003Rosetti et al , 2005. On the other hand, the existence of microheterogeneity on the μm scale range in the surface topography implies the existence of local interactions leading to unfavorable intermolecular mixing of at least some of the components along the lateral plane.…”