Naoshi Ohta

Sphingomyelin levels in the plasma membrane correlate with the activation state of muscle satellite cells

Satellite cells are responsible for postnatal growth, hypertrophy, and regeneration of skeletal muscle. They are normally quiescent, and must be activated to fulfill these functions, yet little is known of how this is regulated. As a first step in determining the role of lipids in this process, we examined the dynamics of sphingomyelin in the plasma membrane. Sphingomyelin contributes to caveolae/lipid rafts, which act to concentrate signaling molecules, and is also a precursor of several bioactive lipids. Proliferating or differentiated C2C12 muscle cells did not bind lysenin, a sphingomyelin-specific binding protein, but noncycling reserve cells did. Quiescent satellite cells also bound lysenin, revealing high levels of sphingomyelin in their plasma membranes. On activation, however, the levels of sphingomyelin drop, so that lysenin did not label proliferating satellite cells. Although most satellite cell progeny differentiate, others stop cycling, maintain Pax7, downregulate MyoD, and escape immediate differentiation. Importantly, many of these Pax7-positive/MyoD-negative cells also regained lysenin binding on their surface, showing that the levels of sphingomyelin had again increased. Our observations show that quiescent satellite cells are characterized by high levels of sphingomyelin in their plasma membranes and that lysenin provides a novel marker of myogenic quiescence.

Biology of lysenin, a protein in the coelomic fluid of the earthworm Eisenia foetida.

Lysenin is a protein of 33?kDa in the coelomic fluid (CF) of the earthworm Eisenia foetida. It differs from other biologically active proteins, such as fetidins, eiseniapore, and coelomic cytolytic factor (CCF-1), that have been found in Eisenia foetida, in terms of both its biochemical and its biological characteristics. The large coelomocytes and free chloragocytes in the typhlosole of Eisenia foetida appear to be the cells that produce lysenin since the mRNA for lysenin and immunoreactive lysenin have been found in these cells. Lysenin binds specifically to sphingomyelin (SM) but not to other phospholipids in cell membranes. After binding to the cell membranes of target cells, lysenin forms oligomers in an SM-dependent manner, with subsequent formation of pores with a hydrodynamic diameter of approximately 3?nm. The biochemical interactions between lysenin and SM in cell membranes are responsible for the pharmacological activities of lysenin and of CF that contains lysenin in vertebrates, such as hemolysis, cytotoxicity, and contraction of smooth muscle in vitro and vasodepressor activity and lethality in vivo. When incubated with SM-liposomes, CF and lysenin lost some or all of their activity, an observation that suggests that SM might be involved in the induction of the various activities of lysenin and CF. However, in general, lysenin is neither cytotoxic nor lethal to invertebrates. An attempt has been made to explain the differences in the responses to lysenin and CF between vertebrates and invertebrates in terms of the presence or absence of SM in the various animals. Among Protostomia, SM is absent in Lophotrochozoa, with the exception of some molluscan species, but it is present in Ecdysozoa, with the exception of Nematomorpha and flies. Among Deuterostomia, Echinodermata and Hemichordata lack SM but SM is found in Chordata. Thus, the difference in terms of the response to lysenin between invertebrates and vertebrates cannot be fully explained by reference to the presence or absence of SM in the organism. Lysenin and its antiserum have made it possible to localize SM in the cell membranes. They should be a useful tool for studies of membrane physiology and the role of SM.

Sites of expression of mRNA for lysenin, a protein isolated from the coelomic fluid of the earthworm Eisenia foetida.

Abstract. 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.

Toxicity of coelomic fluid of the earthworm Eisenia foetida to vertebrates but not invertebrates: probable role of sphingomyelin

The coelomic fluid (CF) of the earthworm Eisenia foetida exhibits a wide variety of biological activities. We found that the CF was not toxic to 42 species, belonging to seven invertebrate phyla, almost all in aquatic adults and larvae exposed to CF. Eleven teleostean species tested died in 0.2-1% CF mostly between 10 and 120 min and the effects were dose-dependent. Tadpoles of the toad Bufo japonicus formosus died in 0.4-2% CF between 80 and 225 min depending upon size, with larger tadpoles surviving longer. Before dying, all experimental tadpoles developed curled and shrunken tails. The Okinawa tree lizard, soft-shelled turtle, Japanese quail, mouse and rat all died after i.v. injection of CF (above 20 microl/kg). Thus, CF was not toxic to invertebrates, but toxic to vertebrates. After heating, CF lost its toxicity to fish, tadpoles and mice. Both CF and lysenin incubated with sphingomyelin-liposomes (SM-liposomes) were no longer toxic, suggesting the involvement of SM in the toxicity. Lysenin, which is a constituent of CF and known to bind specifically to sphingomyelin, exhibited toxicity similar to that of CF. Thus, lysenin in CF is probably responsible for the toxic effects of CF by binding to SM in vertebrate tissues. The bodies of invertebrates might contain little or no SM, while those of vertebrates do contain SM. The coelomic fluid of the earthworm Pheretima communissima has no toxicity to mouse.