Lysenin, a novel 41-kDa
protein purified from coelomic fluid of the earthworm Eisenia foetida, induced erythrocyte lysis. Preincubation of
lysenin with vesicles containing
sphingomyelin inhibited
lysenin-induced
hemolysis completely, whereas vesicles containing
phospholipids other than
sphingomyelin showed no inhibitory activity, suggesting that
lysenin bound specifically to
sphingomyelin on erythrocyte membranes. The specific binding of
lysenin to
sphingomyelin was confirmed by
enzyme-linked
immunosorbent assay, TLC immunostaining, and
liposome lysis assay. In these assays,
lysenin bound specifically to
sphingomyelin and did not show any cross-reaction with other
phospholipids including
sphingomyelin analogs such as
sphingosine,
ceramide, and
sphingosylphosphorylcholine, indicating that it recognized a precise molecular structure of
sphingomyelin. Kinetic analysis of the
lysenin-
sphingomyelin interaction by surface plasmon resonance measurements using BIAcoreTM system showed that
lysenin associated with membrane surfaces composed of
sphingomyelin (kon = 3.2 x 10(4) M-1 s-1) and dissociated extremely slowly (koff = 1.7 x 10(-4) s-1), giving a low dissociation constant (KD = 5.3 x 10(-9) M). Incorporation of
cholesterol into the
sphingomyelin membrane significantly increased the total amount of
lysenin bound to the membrane, whereas it did not change the kinetic parameters of the
lysenin-membrane interaction, suggesting that
lysenin specifically recognized
sphingomyelin and
cholesterol incorporation changed the topological distribution of
sphingomyelin in the membranes, thereby increasing the accessibility of
sphingomyelin to
lysenin. Immunofluorescence staining of fibroblasts derived from a patient with
Niemann-Pick disease type A showed that
lysenin stained the surfaces of the fibroblasts uniformly, whereas intense lysosomal staining was observed when the cells were permeabilized by
digitonin treatment. Preincubation of
lysenin with vesicles containing
sphingomyelin abolished
lysenin immunostaining. This study demonstrated that
lysenin bound specifically to
sphingomyelin on cellular membranes and should be a useful tool to probe the molecular motion and function of
sphingomyelin in
biological membranes.