Binding of a pleurotolysin ortholog from Pleurotus eryngii to sphingomyelin and cholesterol-rich membrane domains

Bhat, Hema Balakrishna; Kishimoto, Tadamitsu; Abe, Mitsuhiro; Makino, Asami; Inaba, Takehiko; Murate, Motohide; Dohmae, Naoshi; Kurahashi, Atsushi; Nishibori, Kozo; Fujimori, Fumihiro; Greimel, Peter; Ishitsuka, Reiko; Kobayashi, Toshihide

doi:10.1194/jlr.d041731

Cited by 48 publications

(65 citation statements)

References 53 publications

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“…C-terminal tryptophan residues, which are well conserved among PlyA, PlyA2, OlyA and EryA, might be responsible for the cholesterol/SM recognition and membrane binding of OlyA-mCherry, as was suggested by tryptophan-to-alanine point mutations of PlyA [28]. Furthermore, in these MDCK cells, OlyA-mCherry was not cytotoxic, as also confirmed for PlyA2-EGFP in HeLa cells and erythrocytes [28], and it did not stimulate or inhibit active signalling pathways that are mediated by tyrosine kinases. However, further studies are needed to fully explore any eventual effects of OlyA-mCherry, and of other aegerolysin-derived fluorescent protein chimeras, on other cell-signalling pathways.…”

Section: Discussionmentioning

confidence: 95%

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Tracking Cholesterol/Sphingomyelin-Rich Membrane Domains with the Ostreolysin A-mCherry Protein

et al. 2014

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Ostreolysin A (OlyA) is an ∼15-kDa protein that has been shown to bind selectively to membranes rich in cholesterol and sphingomyelin. In this study, we investigated whether OlyA fluorescently tagged at the C-terminal with mCherry (OlyA-mCherry) labels cholesterol/sphingomyelin domains in artificial membrane systems and in membranes of Madin-Darby canine kidney (MDCK) epithelial cells. OlyA-mCherry showed similar lipid binding characteristics to non-tagged OlyA. OlyA-mCherry also stained cholesterol/sphingomyelin domains in the plasma membranes of both fixed and living MDCK cells, and in the living cells, this staining was abolished by pretreatment with either methyl-β-cyclodextrin or sphingomyelinase. Double labelling of MDCK cells with OlyA-mCherry and the sphingomyelin-specific markers equinatoxin II–Alexa488 and GST-lysenin, the cholera toxin B subunit as a probe that binds to the ganglioside GM1, or the cholesterol-specific D4 domain of perfringolysin O fused with EGFP, showed different patterns of binding and distribution of OlyA-mCherry in comparison with these other proteins. Furthermore, we show that OlyA-mCherry is internalised in living MDCK cells, and within 90 min it reaches the juxtanuclear region via caveolin-1–positive structures. No binding to membranes could be seen when OlyA-mCherry was expressed in MDCK cells. Altogether, these data clearly indicate that OlyA-mCherry is a promising tool for labelling a distinct pool of cholesterol/sphingomyelin membrane domains in living and fixed cells, and for following these domains when they are apparently internalised by the cell.

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Section: Discussionmentioning

confidence: 95%

“…The closely identical aegerolysin PlyA2 was also recently fused with EGFP and reported to specifically bind cholesterol/SM-rich domains in membranes of HeLa cells [28].…”

Section: Discussionmentioning

confidence: 99%

Tracking Cholesterol/Sphingomyelin-Rich Membrane Domains with the Ostreolysin A-mCherry Protein

et al. 2014

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“…This indicated the importance of the stereochemical configuration of the 3-hydroxyl group on the sterol. Modification of the sterol tail had little influence, while bulky groups on the A-ring and B-ring of the sterol backbone completely abolished PlyA2-EGFP binding (Bhat et al 2013). Membrane binding of Oly was also shown to be dependent on the intact sterol 3β-OH group, and to decrease following minor modifications to the steroid skeleton (Rebolj et al 2006).…”

Section: Aegerolysin Proteins As Membrane Lipid Markersmentioning

confidence: 94%

“…3) (Skočaj et al 2014). Similarly, another almost identical aegerolysin, pleurotolysin A2 (PlyA2), which has also been shown to bind to sphingomyelin-and cholesterolrich membranes, was fused with enhanced green fluorescent protein (EGFP) and used to label fixed HeLa cells (Bhat et al 2013). Pre-incubation of the cells with methyl-β-cyclodextrin or sphingomyelinase totally abolished the binding of both OlyA-mCherry and PlyA2-EGFP to the cells, which demonstrated that both cholesterol and sphingomyelin are prerequisites for the membrane binding of these aegerolysins.…”

Section: Aegerolysin Proteins As Membrane Lipid Markersmentioning

confidence: 99%

Fungal aegerolysin-like proteins: distribution, activities, and applications

Novak

Kraševec

Skočaj

et al. 2014

Appl Microbiol Biotechnol

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The aegerolysin protein family (from aegerolysin of the mushroom Agrocybe aegerita) comprises proteins of ∼15-20 kDa from various eukaryotic and bacterial taxa. Aegerolysins are inconsistently distributed among fungal species, and variable numbers of homologs have been reported for species within the same genus. As such noncore proteins, without a member of a protein family in each of the sequenced fungi, they can give insight into different species-specific processes. Some aegerolysins have been reported to be hemolytically active against mammalian erythrocytes. However, some function as bi-component proteins that have membrane activity in concert with another protein that contains a membrane attack complex/perforin domain. The function of most of aegerolysins is unknown, although some have been suggested to have a role in development of the organism. Potential biotechnological applications of aegerolysins are already evident, despite the limited scientific knowledge available at present. Some mushroom aegerolysins, for example, can be used as markers to detect and label specific membrane lipids. Others can be used as biomarkers of fungal exposure, where their genes can serve as targets for detection of fungi and their progression during infectious diseases. Antibodies against aegerolysins can also be raised as immuno-diagnostic tools. Aegerolysins have been shown to serve as a species determination tool for fungal phytopathogen isolates in terms of some closely related species, where commonly used internal transcribed spacer barcoding has failed. Moreover, strong promoters that regulate aegerolysin genes can promote secretion of heterologous proteins from fungi and have been successfully applied in simultaneous multi-gene expression techniques.

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“…These include cholesterol binding agents such as filippin 85 and Perfringolysin O 86 , sphingolipid reporters such as ostreolysin A 87 , lysenin 88 and pleurotolysin 89 as well as ganglioside lipid ligands such as cholera toxin 90 . The major caveats for these probes are (a) their potential perturbation of native membrane organization, for example by inducing clustering of their binding partners, as is the case for cholera toxin; and (b) their reduced specificity in the cellular context where they can potentially bind off-target species, thereby lowering their specificity for raft domains.…”

Section: Studying Lipid Raftsmentioning

confidence: 99%

The mystery of membrane organization: composition, regulation and roles of lipid rafts

Sezgin

Levental

Mayor

et al. 2017

Nat Rev Mol Cell Biol

1,642

1,700

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Cellular plasma membranes are laterally heterogeneous, featuring a variety of distinct subcompartments that differ in their biophysical properties and composition. A large body of research has focused on understanding the basis for this heterogeneity and its physiological relevance. The membrane raft hypothesis formalized a physicochemical principle for a subtype of such lateral membrane heterogeneity, wherein the preferential associations of cholesterol and saturated lipids drives the formation of relatively packed (ordered) membrane domains that selectively recruit certain lipids and proteins. Recent years have yielded new insights into this concept and its in vivo relevance, primarily owing to the development of biochemical and biophysical technologies.

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Binding of a pleurotolysin ortholog from Pleurotus eryngii to sphingomyelin and cholesterol-rich membrane domains

Cited by 48 publications

References 53 publications

Tracking Cholesterol/Sphingomyelin-Rich Membrane Domains with the Ostreolysin A-mCherry Protein

Tracking Cholesterol/Sphingomyelin-Rich Membrane Domains with the Ostreolysin A-mCherry Protein

Fungal aegerolysin-like proteins: distribution, activities, and applications

The mystery of membrane organization: composition, regulation and roles of lipid rafts

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