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.

Keywords: lipid binding protein; lipid raft; membrane lipids; pore forming toxins; sphingolipid.

Fig. 1.

Isolation and identification of SM/Chol binding protein PlyA2 from P. eryngii. A: Screening of SM/Chol binding protein from P. eryngii. Lane 1, molecular weight (MW) marker. Lane 2, mushroom extract. Lane 3, supernatant fraction after incubation of P. eryngii extract with SM/Chol (1:1) MLVs for 30 min at 37°C followed by sedimentation. Lane 4, pellet fraction after incubation. B: Alignment of amino acid sequence of PlyA2 with PlyA, EryA, and Oly. Protein names are given on the left. Sequence numbers refer to PlyA2 and are shown above. Triangles indicate dissimilar amino acid residues between PlyA and PlyA2. Arrows indicate conserved tryptophan residues. Single letters, amino acids; black background, identical; gray background, conserved substitution.

Fig. 2.

Binding specificity of EGFP-tagged PlyA2 to various liposomes. N-terminally EGFP-tagged PlyA2 (EGFP-PlyA2) (A) or C-terminally EGFP-tagged PlyA2 (PlyA2-EGFP) (B) were incubated with various liposomes followed by centrifugation as described above. The supernatant (S) and pellet (P) fractions were subjected to SDS-PAGE followed by CBB staining. SM/Chol, brain sphingomyelin/cholesterol (1:1) liposomes; POPC/Chol, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/cholesterol (1:1) liposomes; SM/POPC, brain sphingomyelin/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (1:1) liposomes; DPPC/Chol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/cholesterol (1:1) liposomes; PS/Chol, brain phosphatidylserine/cholesterol (1:1) liposomes; GM1/Chol, Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ1-1Cer/cholesterol (1:1) liposomes; GM2/Chol, GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ1-1Cer/cholesterol (1:1) liposomes; GM3/Chol, Neu5Acα2-3Galβ1-4Glcβ1-1Cer/cholesterol (1:1) liposomes; GD1a/Chol, Neu5Acα2-3Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ1-1Cer/cholesterol (1:1) liposomes; LacCer/Chol, Galβ1-4Glcβ1-1Cer/cholesterol (1:1) liposomes; GalCer/Chol, Galβ1-1Cer/cholesterol (1:1) liposomes; SPC/Chol, sphingosylphosphorylcholine/cholesterol (1:1) liposomes; SPH/Chol, sphingosine/cholesterol (1:1) liposomes; Phyto/Chol, D-ribo-phytosphingosine/cholesterol (1:1) liposomes.

Fig. 3.

Binding specificity of PlyA2-EGFP to SM vesicles containing various sterols. A: PlyA2-EGFP was incubated with SM (lane 1), SM/Chol (1:1) (lane 2), SM/β-sitosterol (1:1) (lane 3), SM/lanosterol (1:1) (lane 4), SM/7-dehydrocholesterol (1:1) (lane 5), SM/ergosterol (1:1) (lane 6), SM/6-ketocholestanol (1:1) (lane 7), and SM/epicholesterol (1:1) (lane 8) for 30 min at 37°C. The liposome-bound protein and free protein were separated by a sucrose gradient centrifugation. The top fractions were subjected to SDS-PAGE followed by Western blotting using anti-His antibodies. B: Chemical structure of sterols used in the experiment. Shaded regions indicate the structural difference with Chol.

Fig. 4.

Effects of various treatments on the labeling of HeLa cells with PlyA2-EGFP. Control, PlyA2-EGFP only; SM/Chol and POPC/Chol, PlyA2-EGFP preincubated with indicated liposome mixtures; SMase and MβCD, cells treated with SMase and MβCD, respectively prior to fixation and PlyA2-EGFP labeling. Scale bar, 20 μm.

Fig. 5.

Colocalization of PlyA2-EGFP with lipid raft markers. Cells were fixed and doubly labeled with PlyA2-EGFP and anti-CD59 or anti-GM3. Alternatively, cells were labeled with transferrin followed by fixation and PlyA2-EGFP labeling. Scale bar, 10 μm.

Fig. 6.

Intracellular distribution of PlyA2-EGFP labeling in HeLa cells. Cells were fixed and permeabilized by freezing and thawing. Cells were then doubly labeled with PlyA2-EGFP and various organelle markers. Scale bar, 20 μm.

Fig. 7.

PlyA2-EGFP labels living cells. A: Hemolysis of sheep red blood cells was measured as described in Materials and Methods. B: Living HeLa cells were labeled with exogenously added PlyA2-EGFP. C: PlyA2-AcGFP was expressed in the cytoplasm of HeLa cells as described in Materials and Methods. Scale bar, 20 μm.

Fig. 8.

Tryptophan mutants fail to bind SM/Chol-rich membranes. A: Recombinant PlyA2 or tryptophan mutants were incubated with SM/Chol or PC/Chol liposomes for 30 min at 37°C. Proteins bound to liposomes were recovered by a sucrose gradient centrifugation and detected by Western blotting using anti-His antibodies. B: Fixed HeLa cells were incubated with PlyA2 or tryptophan mutants, followed by treatment with anti-His antibodies and the secondary antibodies conjugated with Alexa 488. Scale bar, 20 μm.

Fig. 9.

Alignment of amino acid sequence of PlyA2 with prominent members of the actinoporin family. Protein names are given on the left. Sequence numbers are indicated above and refer to Eqt2. Choline binding site residues of the actinoporin family are indicated by arrows. Single letters, amino acids; –, alignment gap; black background, identical; gray background, conserved substitution.

Fig. 10.

Cartoon representation of model 2. Position of displayed residues as indicated. N, N terminus; C, C terminus.

Fig. 11.

Putative choline binding site of model 2. A: Model 2. B: Superposition of amino acid residues (as indicated) from Stl2 choline binding site. Color scheme of model 2: carbon (cyan), oxygen (red), and nitrogen (blue). Color scheme of Stl2: carbon (lime), oxygen (pink), nitrogen (iceblue), and phosphorous (purple).