LYSENIN, A NOVEL SPHINGOMYELIN-SPECIFIC BINDING-PROTEIN

Lysenin, a novel 41-kDa protein purified from coelomic fluid of the ea rthworm Eisenia foetida, induced erythrocyte lysis. Preincubation of l ysenin with vesicles containing sphingomyelin inhibited lysenin-induce d hemolysis completely, whereas vesicles containing phospholipids othe r than sphingomyelin showed no inhibitory activity, suggesting that ly senin bound specifically to sphingomyelin on erythrocyte membranes. Th e specific binding of lysenin to sphingomyelin was confirmed by enzyme -linked immunosorbent assay, TLC immunostaining, and liposome lysis as say. In these assays, lysenin bound specifically to sphingomyelin and did not show any cross-reaction with other phospholipids including sph ingomyelin analogs such as sphingosine, ceramide, and sphingosylphosph orylcholine, indicating that it recognized a precise molecular structu re of sphingomyelin. Kinetic analysis of the lysenin-sphingo-myelin in teraction by surface plasmon resonance measurements using BIAcore(TM) system showed that lysenin associated with membrane surfaces composed of sphingomyelin (k(on) = 3.2 x 10(4) M-1 s(-1)) and dissociated extre mely slowly (k(off) = 1.7 x 10(-4) s(-1)), giving a low dissociation c onstant (K-D = 5.3 x 10(-9) M). Incorporation of cholesterol into the sphingomyelin membrane significantly increased the total amount of lys enin bound to the membrane, whereas it did not change the kinetic para meters of the lysenin-membrane interaction, suggesting that lysenin sp ecifically recognized sphingomyelin and cholesterol incorporation chan ged the topological distribution of sphingomyelin in the membranes, th ereby increasing the accessibility of sphingomyelin to lysenin. Immuno fluorescence staining of fibroblasts derived from a patient with Niema nn-Pick disease type A showed that lysenin stained the surfaces of the fibroblasts uniformly, whereas intense lysosomal staining was observe d when the cells were permeabilized by digitonin treatment, Preincubat ion of lysenin with vesicles containing sphingomyelin abolished lyseni n immunostaining. This study demonstrated that lysenin bound specifica lly to sphingomyelin on cellular membranes and should be a useful tool to probe the molecular motion and function of sphingomyelin in biolog ical membranes.