Akiko Yamaji-Hasegawa

A Lipid-Specific Toxin Reveals Heterogeneity of Sphingomyelin-Containing Membranes

Little is known about the heterogenous organization of lipids in biological membranes. Sphingomyelin (SM) is a major plasma membrane lipid that forms lipid domains together with cholesterol and glycolipids. Using SM-specific toxin, lysenin, we showed that in cultured epithelial cells the accessibility of the toxin to SM is different between apical and basolateral membranes. Apical membranes are highly enriched with glycolipids. The inhibitory role of glycolipids in the binding of lysenin to SM was confirmed by comparing the glycolipid-deficient mutant melanoma cell line with its parent cell. Model membrane experiments indicated that glycolipid altered the local density of SM so that the affinity of the lipid for lysenin was decreased. Our results indicate that lysenin recognizes the heterogenous organization of SM in biomembranes and that the organization of SM differs between different cell types and between different membrane domains within the same cell. Isothermal titration calorimetry suggests that lysenin binding to SM is presumably the result of a SM-lysenin complex formation of specific stoichiometry, thus supporting the idea of the existence of small condensed lipid complexes consisting of just a few lipid molecules in living cells.

Total synthesis and biological activities of (+)-sulfamisterin (AB5366) and its analogues.

AbstractThe first total synthesis of (+)-sulfamisterin (AB5366), a naturally occurring α-substituted α-amino acid derivative possessing a sulfonated hydroxy function, is described. Overman rearrangement of an allylic trichloroacetimidate derived from D-tartrate effectively generated the tetrasubstituted carbon containing a nitrogen substituent. Construction of the amino acid moiety and sulfonation of the hydroxy group, followed by deprotection completed the total synthesis, which fully confirmed the proposed absolute structure of the natural product. The possible stereoisomers of (+)-sulfamisterin and their desulfonated derivatives were also synthesized. Biological assessment of all synthetic compounds revealed that natural (+)-sulfamisterin and its 3-epimer as well as their desulfonated derivatives possessing 2S-configuration strongly inhibit the serine palmitoyl transferase both in vitro and in vivo, whereas compounds with 2R-configuration were found to show much weaker inhibitory activity.

Evaluation of aegerolysins as novel tools to detect and visualize ceramide phosphoethanolamine, a major sphingolipid in invertebrates

Ceramide phosphoethanolamine (CPE), a sphingomyelin analog, is a major sphingolipid in invertebrates and parasites, whereas only trace amounts are present in mammalian cells. In this study, mushroom‐derived proteins of the aegerolysin family—pleurotolysin A2 (PlyA2; KD = 12nM), ostreolysin (Oly; KD = 1.3 nM), and erylysin A (EryA; KD = 1.3 nM)—strongly associated with CPE/cholesterol (Chol)‐containing membranes, whereas their low affinity to sphingomyelin/Chol precluded establishment of the binding kinetics. Binding specificity was determined by multilamellar liposome binding assays, supported bilayer assays, and solid‐phase studies against a series of neutral and negatively charged lipid classes mixed 1:1 with Chol or phosphatidylcholine. No cross‐reactivity was detected with phosphatidylethanolamine. Only PlyA2 also associated with CPE, independent of Chol content (KD = 41 μM), rendering it a suitable tool for visualizing CPE in lipid‐blotting experiments and biologic samples from sterol auxotrophic organisms. Visualization of CPE enrichment in the CNS of Drosophila larvae (by PlyA2) and in the bloodstream form of the parasite Trypanosoma brucei (by EryA) by fluorescence imaging demonstrated the versatility of aegerolysin family proteins as efficient tools for detecting and visualizing CPE.—Bhat, H. B., Ishitsuka, R., Inaba, T., Murate, M., Abe, M., Makino, A., Kohyama‐Koganeya, A., Nagao, K., Kurahashi, A., Kishimoto, T., Tahara, M., Yamano, A., Nagamune, K., Hirabayashi, Y., Juni, N., Umeda, M., Fujimori, F., Nishibori, K., Yamaji‐Hasegawa, A., Greimel, P., Kobayashi, T. Evaluation of aegerolysins as novel tools to detect and visualize ceramide phosphoethanolamine, a major sphingolipid in invertebrates. FASEB J. 29, 3920‐3934 (2015). www.fasebj.org

Eudicot plant-specific sphingolipids determine host selectivity of microbial NLP cytolysins

An extra sugar protects Many microbial pathogens produce proteins that are toxic to the cells that they are targeting. Broad-leaved plants are susceptible to NLP (necrosis and ethylene-inducing peptide 1–like protein) toxins. Lenarčič et al. identified the receptors for NLP toxins to be GIPC (glycosylinositol phosphorylceramide) sphingolipids (see the Perspective by Van den Ackerveken). Their findings reveal why these toxins only attack broad-leaved plants (so-called eudicots): If the sphingolipid carries just two hexoses, as is the case for eudicots, the toxin binds and causes cell lysis. But in monocots with sphingolipids that have three hexoses, the toxin is ineffective. Science, this issue p. 1431; see also p. 1383 Plant sensitivity to a microbial cytotoxin is mediated through sugar head groups of an abundant plant sphingolipid. Necrosis and ethylene-inducing peptide 1–like (NLP) proteins constitute a superfamily of proteins produced by plant pathogenic bacteria, fungi, and oomycetes. Many NLPs are cytotoxins that facilitate microbial infection of eudicot, but not of monocot plants. Here, we report glycosylinositol phosphorylceramide (GIPC) sphingolipids as NLP toxin receptors. Plant mutants with altered GIPC composition were more resistant to NLP toxins. Binding studies and x-ray crystallography showed that NLPs form complexes with terminal monomeric hexose moieties of GIPCs that result in conformational changes within the toxin. Insensitivity to NLP cytolysins of monocot plants may be explained by the length of the GIPC head group and the architecture of the NLP sugar-binding site. We unveil early steps in NLP cytolysin action that determine plant clade-specific toxin selectivity.

Pore-forming toxins: Properties, diversity, and uses as tools to image sphingomyelin and ceramide phosphoethanolamine.

Pore-forming toxins (PFTs) represent a unique class of highly specific lipid-binding proteins. The cytotoxicity of these compounds has been overcome through crystallographic structure and mutation studies, facilitating the development of non-toxic lipid probes. As a consequence, non-toxic PFTs have been utilized as highly specific probes to visualize the diversity and dynamics of lipid nanostructures in living and fixed cells. This review is focused on the application of PFTs and their non-toxic analogs as tools to visualize sphingomyelin and ceramide phosphoethanolamine, two major phosphosphingolipids in mammalian and insect cells, respectively. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale.

Molecular mechanisms of action of sphingomyelin-specific pore-forming toxin, lysenin

Lysenin, which is an earthworm toxin, strongly binds to sphingomyelin (SM). Lysenin oligomerizes on SM-rich domains and can induce cell death by forming pores in the membrane. In this review, the assembly of lysenin on SM-containing membranes is discussed mostly on the basis of the information gained by atomic force microscopy (AFM). AFM data show that lysenin assembles into a hexagonal close packed (hcp) structure by rapid reorganization of its oligomers on an SM/cholesterol membrane. In case of a phase-separated membrane of SM, lysenin induces phase mixing as a result of pore formation in SM-rich domains, and consequently its hcp assembly covers the entire membrane. Besides the lytic action, lysenin is important as an SM marker and its pore has the potential to be used as a biosensor in the future. These points are also highlighted in this review.

Intrinsically disordered region of influenza A NP regulates viral genome packaging via interactions with viral RNA and host PI(4,5)P2

To be incorporated into progeny virions, the viral genome must be transported to the inner leaflet of the plasma membrane (PM) and accumulate there. Some viruses utilize lipid components to assemble at the PM. For example, simian virus 40 (SV40) targets the ganglioside GM1 and human immunodeficiency virus type 1 (HIV-1) utilizes phosphatidylinositol (4,5) bisphosphate [PI(4,5)P2]. Recent studies clearly indicate that Rab11-mediated recycling endosomes are required for influenza A virus (IAV) trafficking of vRNPs to the PM but it remains unclear how IAV vRNP localized or accumulate underneath the PM for viral genome incorporation into progeny virions. In this study, we found that the second intrinsically disordered region (IDR2) of NP regulates two binding steps involved in viral genome packaging. First, IDR2 facilitates NP oligomer binding to viral RNA to form vRNP. Secondly, vRNP assemble by interacting with PI(4,5)P2 at the PM via IDR2. These findings suggest that PI(4,5)P2 functions as the determinant of vRNP accumulation at the PM.

Lysenin: A New Probe for Sphingomyelin

Lysenin is a pore-forming toxin that binds to sphingomyelin in a distribution-dependent manner. Studies of this interaction revealed the heterogeneous organization of sphingomyelin in biomembranes while investigations with non-toxic lysenin helped elucidate the spatial and functional heterogeneity of lipid rafts. This chapter summarizes the characterization of lysenin and discusses the possible applications and limitations of this newly developed sphingomyelin probe.