Cholesterol-binding toxins and anti-cholesterol antibodies as structural probes for cholesterol localization

Cholesterol is one of the major constituents of mammalian cell membranes. It plays an indispensable role in regulating the structure and function of cell membranes and affects the pathology of various diseases. In recent decades much attention has been paid to the existence of membrane microdomains, generally termed lipid "rafts", and cholesterol, along with sphingolipids, is thought to play a critical role in raft structural organization and function. Cholesterol-binding probes are likely to provide useful tools for analyzing the distribution and dynamics of membrane cholesterol, as a structural element of raft microdomains, and elsewhere within the cell. Among the probes, non-toxic derivatives of perfringolysin O, a cholesterol-binding cytolysin, bind cholesterol in a concentration-dependent fashion with a strict threshold. They selectively recognize cholesterol in cholesterol-enriched membranes, and have been used in many studies to detect microdomains in plasma and intracellular membranes. Anti-cholesterol antibodies that recognize cholesterol in domain structures have been developed in recent years. In this chapter, we describe the characteristics of these cholesterol-binding proteins and their applications to studies on membrane cholesterol localization.