Sphingomyelinase D Research Articles

Enzymatic studies on planar supported membranes using a widefield fluorescence LAURDAN Generalized Polarization imaging approach

We introduce a custom-built instrument designed to perform fast LAURDAN Generalized Polarization (GP) imaging on planar supported membranes. It is mounted on a widefield fluorescence microscope and allows kinetic analysis of the GP function in the millisecond time scale, largely improving the temporal resolution previously achieved using laser scanning based microscopes. A dedicated protocol to calibrate LAURDAN GP data obtained with charge-coupled device (CCD) cameras as detectors is also presented, enabling reliable assignment of GP values in the field of view. Using this methodology we studied structural and dynamical transformations induced by Sphingomyelinase D (SM-D) on planar supported membranes composed of N-lauroyl sphingomyelin (C12SM). GP data show the evolution of an initially compositionally homogeneous symmetric bilayer existing in a single liquid disordered phase, to an intermediate configuration showing coexistence of liquid disordered and solid ordered domains, which are not always in-register across the axial plane of the bilayer. This intermediate state, caused by the transformation of C12SM to C12-ceramide-1-phosphate in the distal leaflet of the bilayer, evolved to a single solid ordered phase at longer time scales. Additionally, we comparatively studied this system using the membrane fluorophore DiIC18. The advantages and limitations of both fluorescent dyes are discussed, emphasizing the adequacy of LAURDAN GP imaging to explore this type of membrane phenomena.

First report of in vitro selection of RNA aptamers targeted to recombinant Loxosceles laeta spider toxins.

BackgroundLoxoscelism is the envenomation caused by the bite of Loxosceles spp. spiders. It entails severe necrotizing skin lesions, sometimes accompanied by systemic reactions and even death. There are no diagnostic means and treatment is mostly palliative. The main toxin, found in several isoforms in the venom, is sphingomyelinase D (SMD), a phospholipase that has been used to generate antibodies intended for medical applications. Nucleic acid aptamers are a promising alternative to antibodies. Aptamers may be isolated from a combinatorial mixture of oligonucleotides by iterative selection of those that bind to the target. In this work, two Loxosceles laeta SMD isoforms, Ll1 and Ll2, were produced in bacteria and used as targets with the aim of identifying RNA aptamers that inhibit sphingomyelinase activity.ResultsSix RNA aptamers capable of eliciting partial but statistically significant inhibitions of the sphingomyelinase activity of recombinant SMD-Ll1 and SMD-Ll2 were obtained: four aptamers exert ~17% inhibition of SMD-Ll1, while two aptamers result in ~25% inhibition of SMD-Ll2 and ~18% cross inhibition of SMD-Ll1.ConclusionsThis work is the first attempt to obtain aptamers with therapeutic and diagnostic potential for loxoscelism and provides an initial platform to undertake the development of novel anti Loxosceles venom agents.

Exploring Supramolecular Aspects of the Effect of Sphingomyelinase D On Sphingomyelin-Containing Membranes

Lipid-modifying enzymes play a vital role in the regulation of lipids as mediators of cell function. At the same time, the activity of these enzymes is highly affected by the lipid membrane structure. These processes at lipid membranes can be observed in situ through the application of different biophysical techniques. Thus, we are investigating a spider venom enzyme termed sphingomyelinase D (SMD). SMD hydrolyses sphingomyelin (SM) into ceramide-1-phosphate (Cer-1-P). While SM is an integral constituent of many cell membranes, Cer-1-P occurs in very low concentrations and is suggested to be a novel lipid second messenger. At present, the physiologically relevant mechanism following Cer-1-P formation by SMD is incompletely understood, but possibly related to the modulation of membrane properties.Our results show a strong dependency of SMD activity on the phase state of the substrate. SMD is two orders of magnitude more active towards fluid- than gel-phase liposomes. The presence of cholesterol evens out this difference in activity at an intermediate level. The effect of SMD on fluid-phase giant unilamellar vesicles (GUVs) is observed by confocal fluorescence microscopy. GUVs composed of lauroyl-SM show a macroscopic domain formation and/or shrinking and buckling accompanied by the multiple formation of membrane tubes. GUVs composed of egg-SM display a beveling of the membrane and the formation of caps (outside curvature) approx. three days after the addition of SMD. Which membrane morphology evolves is likely a question of enzyme kinetics vs. the dynamics of lipid reorganization.GUVs of raft-like mixtures exhibit a single homogenous phase after the addition of SMD. The consequences of SMD activity and Cer-1-P formation on cellular systems are currently being examined. This will indorse the correlation between enzymatic activity and membrane structure influencing the regulation of physiological processes.

Exploring Molecular and Supramolecular Aspects of Sphingomyelin-Containing Membranes Upon Action of Sphingomyelinase D

Lipid-modifying enzymes play a vital role in the regulation of lipids as mediators of cell function. At the same time, the activity of these enzymes is highly affected by the lipid membrane structure. These processes at lipid membranes can be observed in situ through the application of different biophysical techniques. Thus, we are investigating an enzyme from spider venoms, which is termed sphingomyelinase D (SMD). SMD hydrolyses sphingomyelin (SM) into ceramide-1-phosphate (Cer-1-P). While SM is an integral constituent of many cell membranes, e.g., of red blood cells, Cer-1-P occurs in very low concentrations and is suggested to be a novel lipid second messenger. At present, the physiologically relevant mechanism following Cer-1-P formation by SMD is incompletely understood, but possibly related to the modulation of membrane properties.Our results show a strong dependency of SMD activity on the phase state of the substrate. SMD is one order of magnitude more active towards fluid- than gel-phase liposomes. The effect of SMD on fluid-phase giant unilamellar vesicles (GUVs) is observed by confocal fluorescence microscopy. GUVs composed of lauroyl-SM show a drastic shrinking and buckling accompanied by the multiple formation of membrane tubes, which are up to 80 μm long. Generalized-polarization measurements using the probe LAURDAN exhibit a macroscopic domain formation upon the hydrolysis of lauroyl-SM. This finding points to the induction of membrane curvature by lipid sorting in the simple, binary system of SM/Cer-1-P. SMD activity on GUVs composed of oleoyl-SM also shows tube formation followed by the immediate and complete disintegration of the vesicular membrane structure. The consequences of SMD activity and Cer-1-P formation on cellular systems are currently being examined. This will indorse the correlation between enzymatic activity and membrane structure influencing the regulation of physiological processes.

Membrane structure and the activity of phospholipase and sphingomyelinase D

Lipid-modifying enzymes play a vital role in the regulation of lipids as mediators of cell function. One example is the hydrolysis of phospholipids through phospholipase D (PLD), which produces the signalling molecule phosphatidic acid (PA). These processes at lipid membranes can be observed in situ through the application of different biophysical techniques. Thus, the hydrolysis of phosphatidylcholines by PLD was investigated, showing that the enzyme is highly affected in its catalytic activity by the lipid membrane structure. Briefly, by using Langmuir monolayers as a model system, we revealed that PLD activity depends on the segregation of the hydrolysis product PA within the monolayer. Hence, we could describe how the structure of the PA-rich domains is decisive for the activation and inhibition of PLD. This study demonstrates how membrane structure influences the activity of PLD and regulates the concentration of the lipid messenger PA.The current research project is aiming at describing a toxic component of the venom of brown spiders (Loxosceles), which has a rare enzymatic activity termed sphingomyelinase D (SMD). SMD catalyzes the conversion of sphingomyelin (SM) into ceramide-1-phosphate (Cer-1-P). While the enzymatic substrate SM is an integral constituent of many cell membranes, especially in the vascular epithelium and red blood cells, the reaction product Cer-1-P occurs in very low concentrations. Cer-1-P is suggested to be a novel lipid second messenger in cellular signal transduction events. At present, the precise mechanism of venom action is incompletely understood, but preliminary results show the strong effect of SMD activity on the membrane structure of giant unilamellar vesicles.In summary, the presented work depicts the correlation between membrane structures and the activity of lipid-modifying enzymes. This implements new models for the regulation of cellular processes through distinct structures of biological membranes.

North and South American Loxosceles spiders: Development of a polyvalent antivenom with recombinant sphingomyelinases D as antigens

We report the cloning of sphingomyelinase D (SMD) cDNA from Loxosceles reclusa, Loxosceles boneti and Loxosceles laeta into bacterial expression systems, as well as optimization of expression conditions so as to obtain soluble and active recombinant enzymes. The recombinant mature SMDs, tagged with a histidine tail at the N- or C-termini, were compared in terms of toxicity and enzymatic activity, and were used as immunogens for the production of monovalent antisera in rabbits and F(ab′) 2 preparations in animals used for commercial antivenom production (horses). We performed studies on in vitro inhibition of enzymatic activity of natural venom preparations by antibodies generated against the tagged proteins. We also present and discuss the results of studies on the specific and para-specific in vivo protective potential of the rabbit and equine antibody preparations against the recombinant proteins themselves and natural venom preparations. Our conclusions support the feasibility of using recombinant SMDs for production and evaluation of polyvalent anti- Loxosceles antivenoms, and we offer data on the potential of paraspecific neutralization in the context of the antigenic groupings and the molecular phylogeny of those active SMDs for which amino acid sequence information is available.

The phylogenetic distribution of sphingomyelinase D activity in venoms of Haplogyne spiders

The venoms of Loxosceles spiders cause severe dermonecrotic lesions in human tissues. The venom component sphingomyelinase D (SMD) is a contributor to lesion formation and is unknown elsewhere in the animal kingdom. This study reports comparative analyses of SMD activity and venom composition of select Loxosceles species and representatives of closely related Haplogyne genera. The goal was to identify the phylogenetic group of spiders with SMD and infer the timing of evolutionary origin of this toxin. We also preliminarily characterized variation in molecular masses of venom components in the size range of SMD. SMD activity was detected in all (10) Loxosceles species sampled and two species representing their sister taxon, Sicarius, but not in any other venoms or tissues surveyed. Mass spectrometry analyses indicated that all Loxosceles and Sicarius species surveyed had multiple (at least four to six) molecules in the size range corresponding to known SMD proteins (31–35 kDa), whereas other Haplogynes analyzed had no molecules in this mass range in their venom. This suggests SMD originated in the ancestors of the Loxosceles/Sicarius lineage. These groups of proteins varied in molecular mass across species with North American Loxosceles having 31–32 kDa, African Loxosceles having 32–33.5 kDa and Sicarius having 32–33 kDa molecules.