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J Biol Chem, Vol. 273, Issue 9, 5300-5306, February 27, 1998

Lysenin, a Novel Sphingomyelin-specific Binding Protein

Akiko Yamaji^§, Yoshiyuki Sekizawa^¶, Kazuo Emoto, Hitoshi Sakuraba, Keizo Inoue^§, Hideshi Kobayashi^¶, and Masato Umeda

From the  Department of Inflammation Research and  Department of Clinical Genetics, The Tokyo Metropolitan Institute of Medical Science (Rinshoken), 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113, the ^¶ Research Laboratory, Zenyaku Kogyo Co. Ltd., Nerimaku, Tokyo 178, and the ^§ Department of Health Chemistry, Faculty of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan

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 × 10⁴ M¹ s¹) and dissociated extremely slowly (k_off = 1.7 × 10⁴ s¹), giving a low dissociation constant (K_D = 5.3 × 10⁹ 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.

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