title: Binding of a pleurotolysin ortholog from Pleurotus eryngii to sphingomyelin and cholesterol-rich membrane domains.
abstract: A mixture of sphingomyelin (SM) and cholesterol (Chol) exhibits a characteristic lipid raft domain of the cell membranes that provides a platform to which various signal molecules as well as virus and bacterial proteins are recruited. Several proteins capable of specifically binding either SM or Chol have been reported. However, proteins that selectively bind to SM/Chol mixtures are less well characterized. In our screening for proteins specifically binding to SM/Chol liposomes, we identified a novel ortholog of Pleurotus ostreatus, pleurotolysin (Ply)A, from the extract of edible mushroom Pleurotus eryngii, named PlyA2. Enhanced green fluorescent protein (EGFP)-conjugated PlyA2 bound to SM/Chol but not to phosphatidylcholine/Chol liposomes. Cell surface labeling of PlyA2-EGFP was abolished after sphingomyelinase as well as methyl-β-cyclodextrin treatment, removing SM and Chol, respectively, indicating that PlyA2-EGFP specifically binds cell surface SM/Chol rafts. Tryptophan to alanine point mutation of PlyA2 revealed the importance of C-terminal tryptophan residues for SM/Chol binding. Our results indicate that PlyA2-EGFP is a novel protein probe to label SM/Chol lipid domains both in cell and model membranes.
authors.affiliation: Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan.
authors.forename: Hema Balakrishna
authors.initials: HB
authors.lastname: Bhat
authors.forename: Takuma
authors.initials: T
authors.lastname: Kishimoto
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Abe
authors.forename: Asami
authors.initials: A
authors.lastname: Makino
authors.forename: Takehiko
authors.initials: T
authors.lastname: Inaba
authors.forename: Motohide
authors.initials: M
authors.lastname: Murate
authors.forename: Naoshi
authors.initials: N
authors.lastname: Dohmae
authors.forename: Atsushi
authors.initials: A
authors.lastname: Kurahashi
authors.forename: Kozo
authors.initials: K
authors.lastname: Nishibori
authors.forename: Fumihiro
authors.initials: F
authors.lastname: Fujimori
authors.forename: Peter
authors.initials: P
authors.lastname: Greimel
authors.forename: Reiko
authors.initials: R
authors.lastname: Ishitsuka
authors.forename: Toshihide
authors.initials: T
authors.lastname: Kobayashi
mesh_terms: D000595:Amino Acid Sequence; D002784:Cholesterol / Q000737:chemistry / Q000378:metabolism; D017124:Conserved Sequence; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D006367:HeLa Cells; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D020076:Pleurotus / Q000737:chemistry*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D020539:Sequence Analysis, Protein; D013109:Sphingomyelins / Q000737:chemistry / Q000378:metabolism; D013194:Staining and Labeling
chemical_list: D005656:Fungal Proteins; D006460:Hemolysin Proteins; D011993:Recombinant Fusion Proteins; D013109:Sphingomyelins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D002784:Cholesterol
keywords: lipid binding protein; lipid raft; membrane lipids; pore forming toxins; sphingolipid
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