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 BIAcore TM system showed that lysenin associated with membrane surfaces composed of sphingomyelin (k on ‫؍‬ 3.2 ؋ 10 4 M ؊1 s ؊1 ) and dissociated extremely slowly (k off ‫؍‬ 1.7 ؋ 10 ؊4 s ؊1 ), giving a low dissociation constant (K D ‫؍‬ 5.3 ؋ 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.Sphingomyelin, N-acylsphingosine-1-phosphorylcholine, is a major lipid constituent of animal cell membranes, accounting for up to 60 mol % of the total phospholipid content of some cells, such as sheep erythrocytes (1). In addition to its role as a structural component of membranes, recent studies have suggested the roles of sphingomyelin in various intracellular signaling pathways (2-6). A sphingomyelin cycle has been proposed in which extracellular agents such as tumor necrosis factor ␣, ␥-interferon, and interleukin 1 activate sphingomyelinase, resulting in the production of ceramide which serves as a second messenger mediating the action of these extracellular ligands (4 -6). Sphingomyelin was also proposed to be enriched in an organized membrane domain, caveolae, which contains cholesterol, glyco...

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A novel protein that causes contraction of strips of isolated rat aorta and kills rats upon intravenous injection was purified from the coelomic fluid of the earthworm, Eisenia foetida, by the anion-exchange high-performance liquid chromatography and size-exclusion high-performance liquid chromatography. The protein had a molecular weight of 41 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Contraction of rat aorta was induced at concentrations above l0"9 M. Six partial amino acid sequences of the protein revealed the absence of any significant homology to those of previously characterized proteins, suggesting that the protein is a novel biologically active protein. The protein was named lysenin. In addition, another protein with a molecular weight of 42 kDa, whose amino acid sequence resembled that of lysenin, was also isolated, but it had weak contractive activity.

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Lysenin is a novel protein that was purified from the coelomic fluid of the earthworm Eisenia foetida (Lumbricidae), and it causes contraction of the rat aorta (12). The distribution of this protein was examined immunocytochemically in this species and in the earthworm Pheretima c0m1mm.issima (Megascolecidae) with an antiserum raised in rabbit against lysenin. A positive

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Lysenin is a 33-kDa protein of 297 amino acids that was originally purified from the coelomic fluid of the earthworm Eisenia foetida. It binds specifically to sphingomyelin. In this study, we attempted to identify the site of synthesis of lysenin in the earthworm. We detected the expression of mRNA for lysenin and the presence of immunoreactive lysenin in the large coelomocytes and in the free large chloragocytes present in the lumen of the typhlosole, a depression in the dorsal wall of the intestine. These coelomocytes and chloragocytes seemed to be mature and separate from the chloragogen tissue that lined the typhlosole. The free large chloragocytes in the typhlosole contained numerous vacuoles. The nuclei were small and irregular in shape, and glycogen granules and mitochondria were occasionally found between vacuoles. The chloragocytes of the chloragogen tissue that surrounded the coelomic side of the intestine and the dorsal blood vessel did not react with the lysenin antiserum and no expression of lysenin mRNA was detected in these cells. Furthermore, no evidence of the protein or of the mRNA was found in the cells of the pharyngeal gland. Our findings suggest that lysenin is produced in the free large chloragocytes in the lumen of the typhlosole.