Molecular architecture and functional analysis of NetB, a pore-forming toxin from Clostridium perfringens - PubMed (original) (raw)

In vitro oligomerization and pore-forming activity of NetB. A, NetB oligomerization in the presence of amphiphiles is shown. Monomer and oligomer band positions are indicated. Lane 1, NetB plus 10% (v/v) MPD. Lane 2, NetB plus 20% (v/v) MPD. Lane 3, NetB plus 30% (v/v) MPD. Lane 4, NetB plus 6 m

deoxycholate. Lane 5, NetB plus 12 m

deoxycholate. B, NetB oligomerization in the presence of liposomes is shown. Lane 1, NetB only. Lane 2, NetB plus DPOC:PG (9:1). Lane 3, NetB plus DPOC:PG:Cho (8:1:1). Lane 4, NetB plus DPOC:PG:Cho (7:1:2). Lane 5, NetB plus DPOC:PG:Cho (4:1:5). SDS-PAGE samples were run without boiling. Cho, cholesterol. PE, phosphatidylethanolamine. C, calcein-loaded liposomes of the indicated compositions were incubated with NetB at various concentrations for 1 h at 37 °C, and the % calcein release was measured. D–F, shown are protein lipid overlay assays with NetB (D), C. perfringens enterotoxin (E), and tobacco etch virus protease (F). SphingoStripsTM (Echelon) were incubated with the His6-tagged proteins as described under “Experimental Procedures” and developed using ani-His6 antibodies. Lipid spots: 1, sphingosine; 2, sphingosine-1-phosphate; 3, phytosphingosine; 4, ceramide; 5, sphingomyelin; 6, sphingosylphosphorylcholine; 7, lysophosphatic acid; 8, myriosin; 9, monosialoganglioside; 10, disialoganglioside; 11, 3-sulfogalactoceramide (sulfatide); 12, psychosine; 13, cholesterol; 14, lysophosphocholine; 15, phosphatidylcholine; 16, blank. G, electron microscopy of NetB-liposome mixtures is shown. NetB were mixed with liposomes containing cholesterol (50 mol %). Samples were negatively stained with 2% (w/v) aqueous uranyl acetate and observed using a Philips T12 transmission electron microscope. The liposomes appear completely decorated with 10-nm ring structures, whereas some side views are visible at the liposome edges extending above the liposome surface.