A modified θ-toxin produced by limited proteolysis and methylation: a probe for the functional study of membrane cholesterol

Abstract

A derivative of cytolytic θ-toxin from Clostridium perfringens was prepared by limited proteolytic digestion of the native toxin followed by methylation. Among the chloroform/methanol-extractable, lipid components of sheep and human erythrocytes, the proteinase-nicked and methylated derivative (MCθ) specifically binds to cholesterol. While MCθ retains binding affinity comparable to that of intact toxin, it causes no obvious membrane damage, resulting in no hemolysis at temperatures of 37° C or lower. Using MCθ, we demonstrated the possible existence of high- and low-affinity sites for θ-toxin on sheep erythrocytes at both 37° C and 10° C. The number of high-affinity sites on sheep erythrocytes was estimated to be approximately 3-times larger at 37° C than that at 10° C. In addition, high- and low-affinity sites were demonstrated in human erythrocytes and a lymphoma B cell line, BALL-1 cells. Both binding sites disappear upon simultaneous treatment of cells with sublytic doses of digitonin, suggesting that cholesterol is an essential component of both the high- and low-affinity sites and that the mode of cholesterol existence in plasma membranes is heterogeneous in these cells. Because of its high affinity for membrane cholesterol without causing any obvious membrane changes at physiological temperatures, MCθ may provide a probe for use in the functional study of membrane cholesterol.