Biophysics of sphingolipids II. Glycosphingolipids: an assortment of multiple structural information transducers at the membrane surface.

Glycosphingolipids are ubiquitous components of animal cell membranes. They are constituted by the basic structure of ceramide with its hydroxyl group linked to single carbohydrates or oligosaccharide chains of different complexity. The combination of the properties of their hydrocarbon moiety with those derived from the variety and complexity of their hydrophilic polar head groups confers to these lipids an extraordinary capacity for molecular-to-supramolecular transduction across the lateral/transverse planes in biomembranes and beyond. In our opinion, most of the advances made over the last decade on the biophysical behavior of glycosphingolipids can be organized into three related aspects of increasing structural complexity: (1) intrinsic codes: local molecular interactions of glycosphingolipids translated into structural self-organization. (2) Surface topography: projection of molecular shape and miscibility of glycosphingolipids into formation of coexisting membrane domains. (3) Beyond the membrane interface: glycosphingolipid as modulators of structural topology, bilayer recombination and surface biocatalysis.

Protein-mediated surface structuring in biomembranes

The lipids and proteins of biomembranes exhibit highly dissimilar conformations, geometrical shapes, amphipathicity, and thermodynamic properties which constrain their two-dimensional molecular packing, electrostatics, and interaction preferences. This causes inevitable development of large local tensions that frequently relax into phase or compositional immiscibility along lateral and transverse planes of the membrane. On the other hand, these effects constitute the very codes that mediate molecular and structural changes determining and controlling the possibilities for enzymatic activity, apposition and recombination in biomembranes. The presence of proteins constitutes a major perturbing factor for the membrane sculpturing both in terms of its surface topography and dynamics. We will focus on some results from our group within this context and summarize some recent evidence for the active involvement of extrinsic (myelin basic protein), integral (Folch-Lees proteolipid protein) and amphitropic (c-Fos and c-Jun) proteins, as well as a membrane-active amphitropic phosphohydrolytic enzyme (neutral sphingomyelinase), in the process of lateral segregation and dynamics of phase domains, sculpturing of the surface topography, and the bi-directional modulation of the membrane biochemical reactivity.

Protein-mediated surface structuring in biomembranes.

The lipids and proteins of biomembranes exhibit highly dissimilar conformations, geometrical shapes, amphipathicity, and thermodynamic properties which constrain their two-dimensional molecular packing, electrostatics, and interaction preferences. This causes inevitable development of large local tensions that frequently relax into phase or compositional immiscibility along lateral and transverse planes of the membrane. On the other hand, these effects constitute the very codes that mediate molecular and structural changes determining and controlling the possibilities for enzymatic activity, apposition and recombination in biomembranes. The presence of proteins constitutes a major perturbing factor for the membrane sculpturing both in terms of its surface topography and dynamics. We will focus on some results from our group within this context and summarize some recent evidence for the active involvement of extrinsic (myelin basic protein), integral (Folch-Lees proteolipid protein) and amphitropic (c-Fos and c-Jun) proteins, as well as a membrane-active amphitropic phosphohydrolytic enzyme (neutral sphingomyelinase), in the process of lateral segregation and dynamics of phase domains, sculpturing of the surface topography, and the bi-directional modulation of the membrane biochemical reactivity.

Kinetic steps for the hydrolysis of sphingomyelin by Bacillus cereus sphingomyelinase in lipid monolayers.

The sphingomyelinase (Sphmase) activity degrading sphingomyelin (Sphm) monolayers shows a slow-reaction latency period before exhibiting constant rate catalysis. These two kinetic regions are regulated independently by the lateral surface pressure and by lipids that are biomodulators of cell function such as ceramide, glycosphingolipids, fatty acids, and lysophospholipids. Knowledge of the interfacial adsorption of Sphmase, precatalytic activation, initiation of effective catalysis, and the corresponding kinetic parameters is necessary for studying the level at which different lipids modulate the activity. We dissected some kinetic steps and determined the rate constants for degradation of Sphm, under controlled intermolecular organization, by Sphmase. Six models, adapted to two dimensions, were used to elucidate possible mechanisms for the interfacial activation of Sphmase during the lag time. The models consider enzyme binding to the substrate monolayer and a subsequent, essentially irreversible interfacial activation; this is supported experimentally by monolayer transfer experiments. Some mechanisms involve enzyme-substrate binding and associated states of the enzyme in the bulk subphase or at the interface, prior to complete activation. The activity of Sphmase is consistent with kinetics involving enzyme partitioning into the interface followed by substrate association, and by a process that proceeds with bimolecular kinetic dependence on the interfacial Sphmase concentration, and a subsequent slow step of activation. A possible equilibrium between the apparent monomolecular and bimolecular activated states of the interfacial enzyme, coupled to a slow activation, constitute rate-limiting steps that can explain the existence of lag time and the achievement of a maximum constant rate of degradation of Sphm monolayers by Sphmase.

Surface pressure-dependent cross-modulation of sphingomyelinase and phospholipase A2 in monolayers.

We investigated the ways in which phospholipase A2 and sphingomyelinase are mutually modulated at lipid interfaces. The activity of one enzyme is affected by its own reaction products and by substrates and products of the other enzyme; all this depends differently on the lateral surface pressure. Ceramide inhibits both the sphingomyelinase activity rate and the extent of degradation, and decreases the lag time at all surface pressures. Dilauroyl- and dipalmitoylphosphatidylcholine, the substrates of phospholipase A2 (PLA2), do not affect sphingomyelinase activity. The products of PLA2, palmitic acid and lysopalmitoylphosphatidylcholine, strongly enhance and shift to high surface pressures the activity optimum and the cutoff point of sphingomyelinase. Palmitic acid also shifts to high surface pressures the cut-off point of PLA2 activity. Sphingomyelin strongly inhibits PLA2 at surface pressures above 5 mN/m, while ceramide shifts the cut-off point and the activity optimum to high surface pressures. The sphingolipids increase the lag time of PLA2 at low surface pressures. Both phosphohydrolytic pathways involve different levels of control on precatalytic steps and on the rate of activity that appear independent on specific alterations of molecular packing and surface potential. The mutual lipid-mediated interfacial modulation between both phosphohydrolytic pathways indicates that phospholipid degradation may be self-amplified or dampened depending on subtle changes of surface pressure and composition.

Mutual modulation of sphingomyelinase and phospholipase A2 activities against mixed lipid monolayers by their lipid intermediates and glycosphingolipids.

Sphingomyelinase activity against pure sphingomyelin monolayers is constant up to a surface pressure of 18 mN/m and falls above it. Sphingomyelinase- and phospholipase A2-mediated phosphohydrolytic pathways are mutually modulated by the presence of their respective substrates and products. At 15 mN/m non-substrate lipids such as ceramide at a mole fraction of 0.1 in mixed films with the pure substrate, inhibit the sphingomyelinase activity. Ganglioside GM1, another ceramide-containing complex sphingolipid, also inhibits sphingomyelinase activity, while a chemically related glycosphingolipid such as asialo-GM1 has no effect. The activity is unaltered by dipalmitoylphosphatidylcholine and by an equimolar mixture of its products of hydrolysis by phospholipase A2, fatty acid and lysoderivative, but it is inhibited by only one of them or by dilauroylphosphatidylcholine. Phospholipase A2 is inhibited by sphingomyelin, and activated by ceramide and by palmitic acid, one of the products of its own phosphohydrolytic reaction.