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

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

doi:10.1194/jlr.d041731

Hema Balakrishna Bhat, Takuma Kishimoto + Show 11 more

Open Access

https://doi.org/10.1194/jlr.d041731

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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.

Highlights

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
We identified a novel ortholog of PlyA, termed PlyA2, from the extracts of P. eryngii, capable of binding to SM/Chol liposomes
The following were purchased from Avanti Polar Lipids (Alabaster, AL): SM; L-␣-phosphatidylcholine (PC); 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC); DPPC; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (N-NBD-PE); 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE); L-␣-phosphatidylserine (PS); lactosyl ceramide (LacCer) (Gal␤1-4Glc␤1-1Cer); and galactosyl ceramide (GalCer) (Gal␤1-1Cer)

Summary

Introduction

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. Our results indicate that PlyA2EGFP is a novel protein probe to label SM/Chol lipid domains both in cell and model membranes.—Bhat, H. Together with cholesterol (Chol), SM forms specific lipid microdomains in both model [1] and biomembranes [2]. These domains are designated as lipid rafts [3,4,5].

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