Sphingomyelinase acts by an area-activated mechanism on the liquid-expanded phase of sphingomyelin monolayers

Abstract

We describe the localization of Alexa-488-labeled SMase in SM/ceramide (Cer) lipid monolayers containing segregated liquid-condensed (LC) Cer-enriched domains surrounded by a continuous liquid-expanded (LE) SMenriched phase. Langmuir-Schaefer films were made in order to visualize the labeled enzyme. Independently of initial conditions Alexa-SMase is preferably localized in the SMenriched LE phase and it is not enriched at the domain boundaries. A novel mechanism is proposed for the action of SMase, which can also explain the regulatory effect of the surface topography on the enzyme activity. The homogeneous enzymatic generation of Cer in the LE phase leads to a meta-stable, kinetically trapped, supersaturated mixed monolayer. This effect acts as driving force for the segregation of the Cer-enriched domain following classical nucleation mechanisms. Accordingly, the number and size of Cer-enriched domains are determined by the extent of Cer supersaturation in the LE phase rather than by the SMase local activity. The kinetic barrier for nucleation, for which a compositional gap of at least 53 mol% of Cer is necessary to reach a thermodynamically stable LC phase, can explain the lag time to reaching full catalytic activity. Altogether, the data support an "area-activated mechanism," in which the enzyme is homogeneously active over the LE surface. Phospholipases are a group of mostly water-soluble enzymes, widely spread in nature, that perform their catalytic activity at an interface, owing to the insoluble nature of their substrates (1). Several studies have equated "membrane defects," such as those arising from the coexistence of lipid domains in different physical states, with enhanced catalytic activity (2-8). In 1990, it was reported that the liquidexpanded (LE), liquid-condensed (LC) lateral interfaces (lipid domain borders) in a one-component monolayer of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) act as starting points for Naja naja phospholipase A 2 (PLA 2 ) catalytic activity (9). Dahmen-Levison, Brezesinski, and Mohwald (10) showed in lipid monolayers that the fluorescent-labeled PLA 2 preferably accumulates at the LC-LE interface of domains. Those results support the socalled perimeter-activated or border-activated mechanism, in which the enzyme must have physical contact with the domain boundary/membrane defect in order to become fully active and exerts its major catalytic action adsorbed to these linear interfaces (11). However, more recent studies (12) have demonstrated that fluorescein-labeled PLA 2 from Crotalus atrox was homogeneously distributed in 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and DPPC giant unilamellar vesicles when the lipids were in the LE state and preferentially localized in the LE phase at temperatures corresponding to the gel-fluid phase coexistence. The homogeneous distribution of PLA 2 on the membrane surface would support a mechanism by which the enzyme is fully active over the whole fluid surface (area-activated mechanism) but cannot adequately explain...

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

“…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).…”

Ceramide-enriched microdomains in planar membranes

“…Maggio, Fanani and coworkers have reported detailed fluorescence microscopy studies of the enzymatic generation of Cer in SM monolayers in the liquid-expanded (LE) phase at the air-water interface [9,51,[54][55][56]. Their work represents one of the most comprehensive examinations of the factors that influence membrane reorganization during and after SMase activity.…”

“…Several aspects of the SMase hydrolysis of SM monolayers suggest that enzymatic reaction leads to monolayer morphologies that are far from equilibrium [54][55][56]. These include the observation of different shapes and sizes for Cer-enriched domains prepared by direct mixing and enzyme action and the formation of a supersaturated Cerenriched phase during the initial lag period.…”

“…Although a border-activated mechanism has been hypothesized for a number of phospholipases, other evidence suggests that some phospholipases are active over the entire fluid surface of a membrane, rather than at domain boundaries (an excellent literature summary is provided in a recent review [9]). To probe which mechanism applies to SMase hydrolysis of SM monolayers, the distribution of Alexa-488-labeled SMase was examined on SM and 9:1 SM/Cer monolayers at various stages of enzyme activity [55]. SMase was found to localize preferentially in the LE phase of both SM and SM/Cer monolayers, with no evidence for higher enzyme concentration at domain boundaries.…”

“…The monolayer studies described above illustrate the complex interplay between monolayer morphology and enzyme activity and raise several mechanistic questions that have been addressed during the last two years [54][55][56]. The correlation between the formation of Cer-enriched domains and the development of full catalytic capacity at the end of the lag period was examined [56].…”