Epidemiological, clinical and biochemical characterization of the p.(Ala359Asp) SMPD1 variant causing Niemann-Pick disease type B.

Niemann-Pick disease type B (NPDB) is a rare, inherited lysosomal storage disorder that occurs due to variants in the sphingomyelin phosphodiesterase 1 (SMPD1) gene and the resultant deficiency of acid sphingomyelinase (ASM) activity. While numerous variants causing NPDB have been described, only a small number have been studied in any detail. Herein, we describe the frequency of the p.(Ala359Asp) variant in the healthy Chilean population, and determine the haplotype background of homozygous patients to establish if this variant originated from a common founder. Genomic DNA samples from 1691 healthy individuals were analyzed for the p.(Ala359Asp) variant. The frequency of p.(Ala359Asp) was found to be 1/105.7, predicting a disease incidence of 1/44 960 in Chile, higher than the incidence estimated by the number of confirmed NPDB cases. We also describe the clinical characteristics of 13 patients homozygous for p.(Ala359Asp) and all of them had moderate to severe NPDB disease. In addition, a conserved haplotype and shared 280 Kb region around the SMPD1 gene was observed in the patients analyzed, indicating that the variant originated from a common ancestor. The haplotype frequency and mitochondrial DNA analysis suggest an Amerindian origin for the variant. To assess the effect of the p.(Ala359Asp) variant, we transfected cells with the ASM-p.(Ala359Asp) cDNA and the activity was only 4.2% compared with the wild-type cDNA, definitively demonstrating the causative effect of the variant on ASM function. Information on common variants such as p.(Ala359Asp) is essential to guide the successful implementation for future therapies and benefit to patients.

Generation and characterization of a recombinant chimeric protein (rCpLi) consisting of B-cell epitopes of a dermonecrotic protein from Loxosceles intermedia spider venom.

A chimeric protein was constructed expressing three epitopes of LiD1, a dermonecrotic toxin from the venom of Loxosceles intermedia spider. This species is responsible for a large number of accidents involving spiders in Brazil. We demonstrated that the chimeric protein (rCpLi) generated is atoxic and that antibodies previously developed in rabbits against synthetic epitopes reactive with rCpLi in ELISA and immunoblot assays. The antibody response in rabbits against the rCpLi was evaluated by ELISA and we have detected an antibody response in all immunized animals. Overlapping peptides covering the amino acid sequence of the rCpLi were synthesized on a cellulose membrane, and their recognition by rabbit anti-rCpLi serum assessed. Three different antigenic regions were identified. The percentage of inhibition of the dermonecrotic, hemorrhagic and edematogenic activities caused by the recombinant protein LiD1r in naïve rabbits was assessed by pre-incubation with anti-rCpLi antibodies. Anti-rCpLi induced good dermonecrotic and hemorrhagic protection. The levels of protection were similar to the antiboides anti-LiD1r. In summary, we have developed a polyepitope recombinant chimeric protein capable of inducing multiple responses of neutralizing antibodies in a rabbit model. This engineered protein may be a promising candidate for therapeutic serum development or vaccination.

Ordered-disordered domain coexistence in ternary lipid monolayers activates sphingomyelinase by clearing ceramide from the active phase.

We explored the action of sphingomyelinase (SMase) on ternary monolayers containing phosphatidylcholine, sphingomyelin (SM) and dihydrocholesterol, which varied along a single tie line of phase coexistence. SMase activity exhibited a higher rate and extent of hydrolysis when the film is within the liquid-expanded (LE)/liquid-ordered (LO) coexistence range, compared to monolayers in the full LO phase. Since Alexa-SMase preferably adsorbs to the LE phase and there was no direct correlation found between enzymatic activity and domain borders, we postulate that the LE phase is the active phase for ceramide (Cer) generation. The enzymatically generated Cer was organized in different ways depending on the initial LE/LO ratio. The action of SMase in Chol-poor monolayers led to the formation of Cer-enriched domains, while in Chol-rich monolayers it resulted in the incorporation of Cer in the LO phase and the formation of new Chol- and Cer-enriched domains. The following novel mechanism is proposed to provide an explanation for the favored action of SMase on interfaces that exhibit an LE-LO phase coexistence: the LO phase sequesters the product Cer causing its depletion from the more enzyme-susceptible LE phase, thus decreasing inhibition by the reaction product. Furthermore, LO domains function as a substrate reservoir by allowing a rapid exchange of the substrate from this phase to the SM-depleted LE phase.

The action of sphingomyelinase in lipid monolayers as revealed by microscopic image analysis.

In recent years, new evidence in biomembrane research brought about a holistic, supramolecular view on membrane-mediated signal transduction. The consequences of sphingomyelinase (SMase)-driven formation of ceramide (Cer) at the membrane interface involves reorganization of the lateral membrane structure of lipids and proteins from the nm to the mum level. In this review, we present recent insights about mechanisms and features of the SMase-mediated formation of Cer-enriched domains in model membranes, which have been elucidated through a combination of microscopic techniques with advanced image processing algorithms. This approach extracts subtle morphological and pattern information beyond the visual perception: since domain patterns are the consequences of subjacent biophysical properties, a reliable quantitative description of the supramolecular structure of the membrane domains yields a direct readout of biophysical properties which are difficult to determine otherwise. Most of the information about SMase action on simple lipid interfaces has arisen from monolayer studies, but the correspondence to lipid bilayer systems will also be discussed. Furthermore, the structural changes induced by sphingomyelinase action are not fully explained just by the presence of ceramide but by out-of equilibrium surface dynamics forcing the lipid domains to adopt transient supramolecular pattern with explicit interaction potentials. This rearrangement responds to a few basic physical properties like lipid mixing/demixing kinetics, electrostatic repulsion and line tension. The possible implications of such transient codes for signal transduction are discussed for SMase controlled action on lipid interfaces.

Sphingomyelinase-induced domain shape relaxation driven by out-of-equilibrium changes of composition.

Sphingomyelinase (SMase)-induced ceramide (Cer)-enriched domains in a lipid monolayer are shown to result from an out-of-equilibrium situation. This is induced by a change of composition caused by the enzymatic production of Cer in a sphingomyelin (SM) monolayer that leads to a fast SM/Cer demixing into a liquid-condensed (LC), Cer-enriched and a liquid-expanded, SM-enriched phases. The morphological evolution and kinetic dependence of Cer-enriched domains is studied under continuous observation by epifluorescence microscopy. Domain shape annealing is observed from branched to rounded shapes after SMase activity quenching by EDTA, with a decay halftime of approximately 10 min. An out-of-equilibrium fast domain growth is not the determinant factor for domain morphology. Domain shape rearrangement in nearly equilibrium conditions result from the counteraction of intradomain dipolar repulsion and line tension, according to McConnell's shape transition theory. Phase separation causes a transient compositional overshoot within the LC phase that implies an increased out-of-equilibrium enrichment of Cer into the LC domains. As a consequence, higher intradomain repulsion leads to transient branched structures that relax to rounded shapes by lowering the proportion of Cer in the domain to equilibrium values. The fast action of SMase can be taken as a compositional perturbation that brings about important consequences for the surface organization.

Conformational changes of Loxosceles venom sphingomyelinases monitored by circular dichroism.

Envenomation by arachnids of the genus Loxosceles can induce a variety of biological effects, including dermonecrosis and hemolysis. We have previously identified in L. intermedia venom two highly homologous proteins with sphingomyelinase activity, termed P1 and P2, responsible for all these pathological events, and also an inactive isoform P3. The toxins P1 and P2 displayed 85% identity with each other at the amino acid level and showed a 57% identity with SMase I, an active toxin from L. laeta venom. Circular dichroism was used to determine and compare the solution structure of the active and inactive isoforms. Effects of pH and temperature change on the CD spectra of the toxins were investigated and correlated with the biological activities. This study sheds new light on the structure-function relationship of homologous proteins with distinct biological properties and represents the first report on the structure-function relationship of Loxosceles sphingomyelinases D.

Shape transitions and lattice structuring of ceramide-enriched domains generated by sphingomyelinase in lipid monolayers.

Sphingomyelinases (SMases) hydrolyze the membrane constituent sphingomyelin (SM) to phosphocholine and ceramide (Cer). Growing evidence supports that SMase-induced SM-->Cer conversion leads to the formation of lateral Cer-enriched domains which drive structural reorganization in lipid membranes. We previously provided visual evidence in real-time for the formation of Cer-enriched domains in SM monolayers through the action of the neutral Bacillus cereus SMase. In this work, we disclose a succession of discrete morphologic transitions and lateral organization of Cer-enriched domains that underlay the SMase-generated surface topography. We further reveal how these structural parameters couple to the generation of two-dimensional electrostatic fields, based upon the specific orientation of the lipid dipole moments in the Cer-enriched domains. Advanced image processing routines in combination with time-resolved epifluorescence microscopy on Langmuir monolayers revealed: 1), spontaneous nucleation and circular growth of Cer-enriched domains after injection of SMase into the subphase of the SM monolayer; 2), domain-intrinsic discrete transitions from circular to periodically undulating shapes followed by a second transition toward increasingly branched morphologies; 3), lateral superstructure organization into predominantly hexagonal domain lattices; 4), formation of super-superstructures by the hexagonal lattices; and 5), rotationally and laterally coupled domain movement before domain border contact. All patterns proved to be specific for the SMase-driven system since they could not be observed with Cer-enriched domains generated by defined mixtures of SM/Cer in enzyme-free monolayers at the same surface pressure (pi = 10 mN/m). Following the theories of lateral shape transitions, dipolar electrostatic interactions of lipid domains, and direct determinations of the monolayer dipole potential, our data show that SMase induces a domain-specific packing and orientation of the molecular dipole moments perpendicular to the air/water interface. In consequence, protein-driven generation of specific out-of-equilibrium states, an accepted concept for maintenance of transmembrane lipid asymmetry, must also be considered on the lateral level. Lateral enzyme-specific out-of-equilibrium organization of lipid domains represents a new level of signal transduction from local (nm) to long-range (microm) scales. The cross-talk between lateral domain structures and dipolar electrostatic fields adds new perspectives to the mechanisms of SMase-mediated signal transduction in biological membranes.

Crystallization and preliminary crystallographic analysis of SMase I, a sphingomyelinase from Loxosceles laeta spider venom.

SMase I, a 32 kDa sphingomyelinase found in Loxosceles laeta venom, is responsible for the major pathological effects of spider envenomation. This toxin has been cloned and functionally expressed as a fusion protein containing a 6 x His tag at its N-terminus to yield a 33 kDa protein [Fernandes-Pedrosa et al. (2002), Biochem. Biophys. Res. Commun. 298, 638-645]. The recombinant protein possesses all the biological properties ascribed to the whole L. laeta venom, including dermonecrotic and complement-dependent haemolytic activities. Dynamic light-scattering experiments conducted at 291 K demonstrate that the sample possesses a monomodal distribution, with a hydrodynamic radius of 3.57 nm. L. laeta SMase I was crystallized by the hanging-drop vapour-diffusion technique using the sparse-matrix method. Single crystals were obtained using a buffer solution consisting of 0.08 M HEPES and 0.9 M trisodium citrate, which was titrated to pH 7.5 using 0.25 M sodium hydroxide. Complete three-dimensional diffraction data were collected to 1.8 angstroms at the Laboratório Nacional de Luz Síncrotron (LNLS, Campinas, Brazil). The crystals belong to the hexagonal system (space group P6(1) or P6(5)), with unit-cell parameters a = b = 140.6, c = 113.6 angstroms. A search for heavy-atom derivatives has been initiated and elucidation of the crystal structure is currently in progress.

Bidirectional control of sphingomyelinase activity and surface topography in lipid monolayers.

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.

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.

Sphingomyelinases in the venom of the spider Loxosceles intermedia are responsible for both dermonecrosis and complement-dependent hemolysis.

The bite of spiders of the genus Loxosceles can induce a variety of biological effects, including dermonecrosis and complement (C) dependent haemolysis. The aim of this study was to characterise the toxins in the venom responsible for the different biological effects. We have previously shown that a 35 kDa protein, named F35, purified from Loxosceles intermedia venom, incorporates into the membranes of human erythrocytes and renders them susceptible to the alternative pathway of autologous C. Here we have further purified the F35 protein which was resolved by reversed phase chromatography into three tightly contiguous peaks termed P1, P2, and P3. P1 and P2 were shown to be homogeneous by SDS-PAGE and N-terminal aminoacid analysis, while P3 consisted of two highly homologous proteins. N-terminal sequencing of all four proteins showed a high degree of homology, which was confirmed by cross-reactivity of antisera raised against the individual purified proteins. Functional characterisation of P1 and P2 indicated the presence of sphingomyelinase activity and either protein in isolation was capable of inducing all the in vivo effects seen with whole spider venom, including C-dependent haemolysis and dermonecrosis. In all assays, P2 was more active than P1, while P3 was completely inactive. These data show that different biological effects of L. intermedia venom can be assigned to the sphingomyelinase activity of two highly homologous proteins, P1 and P2. Identification of these proteins as inducers of the principal pathological effects induced by whole venom will aid studies of the mechanism of action of the venom and the development of a effective therapy.

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.