Purification, molecular cloning, and application of a novel sphingomyelin-binding protein (clamlysin) from the brackishwater clam, Corbicula japonica Taketoshi Takara a , Tetsuto Nakagawa a,1 , Masami Isobe a , Nozomu Okino a , Sachiyo Ichinose b , Akira Omori b , Makoto Ito a, ⁎ a Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan b Mitsubishi Kagaku Institute of Life Sciences, 11 Minamiooya, Machida 194-8511, Tokyo, Japan abstract article info Article history: Received 6 October 2010 Received in revised form 10 February 2011 Accepted 22 February 2011 Available online 8 March 2011 Keywords: Sphingomyelin Hemolytic protein Actinoporin family Clamlysin A novel sphingomyelin-binding protein (clamlysin) was purified from the foot muscle of a brackishwater clam, Corbicula japonica. The purified 24.8-kDa protein lysed sheep, horse and rabbit erythrocytes and the hemolytic activity was inhibited by sphingomyelin, but not other phospholipids or glycosphingolipids. The open reading frame of the clamlysin gene encoded a putative 26.9-kDa protein (clamlysin B) which showed high sequence similarity with the actinoporin family. A surface plasmon resonance assay confirmed that clamlysin B specifically bound to sphingomyelin. Furthermore, two cDNA variants of clamlysin, encoding putative 31.4 kDa (clamlysin A) and 11 kDa (clamlysin C) proteins, were isolated. Only the 31.4-kDa variant was found to exhibit sphingomyelin-binding activity. Clamlysin A and B, but not C, shared a sequence (domain II) conserved in all known sphingomyelin-binding proteins. Domain II fused with a glutathione S-transferase bound to sphingomyelin. Horse erythrocytes, mouse melanoma B16 and GM95 cells, and Chinese hamster ovary CHO-K1 cells, but not the same cells treated with bacterial sphingomyelinase, were immunostained with clamlysin B. These results indicate that clamlysin B binds to the sphingomyelin of living cells and thus would be useful as a molecular probe to detect sphingomyelin. © 2011 Elsevier B.V. All rights reserved. 1. Introduction The cytolytic and hemolytic proteins produced by many pathogenic bacteria, such as alpha-toxin [1], leukocidin [2], and perfringolysin O [3], are considered to act as effectors or toxins during or after an infection. On the other hand, some invertebrates produce pore- forming toxins which are conceivably utilized for self-defense against predators and/or capturing prey [4,5]. The basic 20-kDa toxins in sea anemone venom, highly conserved proteins with high sequence similarity, were named actinoporins [5]. Recently, proteins showing similarity to actinoporins have been found in not only sea anemones but also three animal and two plant phyla, and named AF (actinoporin- like proteins and fungal fruit-body lectins) domain superfamily proteins [6]. Actinoporins were shown to be lethal to crustaceans and fish that are possible prey for sea anemones [5], while the fungal lectins belonging to the AF superfamily showed insecticidal and anti- proliferative actions toward some insects and cultured cells [7,8]. However, the true physiological roles of the AF superfamily including actinoporins have yet to be fully elucidated. Interestingly, actinoporins make pores in lipid membranes containing sphingomyelin (SM), whereas the fungal lectins specifically bind to T-antigen (Galβ1-3GalNAc) on some malignant cells [9]. An orthologue of zebrafish, Dr1, exhibited the membrane-binding activity but did not show the SM specificity [6]. It is worth noting that the secondary structure of these proteins is remarkably similar regardless of the different binding specificity [6]. The structure of equinatoxin II [10] and sticholysin II [11], representatives of actinoporin family proteins, has been solved. They are extremely similar in structure and composed of a tightly folded β-sandwich core flanked on two sides by an α-helix, one of which is located at the N-terminal and might participate in the toxin's penetration of the lipid membrane. Interestingly, the α-helix in the N-terminal region is absent in the fungal lectins [12]. The crystal structure of sticholysin II with phosphocholine, the common head group of SM and phosphatidylcholine (PC), was solved and the binding site for phosphocholine in actinoporins was determined [11]. However, the precise mechanism for binding to SM remains to be solved, because no structural details concerning a complex of the protein with SM are available. Biochimica et Biophysica Acta 1811 (2011) 323–332 Abbreviations: Chol, cholesterol; DLPC, dilauroylphosphatidylcholine; GalCer, galactosylceramide; GM1, monosialylgangliotetraosylceramide; GST, glutathione S-transferase; SM, sphingomyelin; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine ⁎ Corresponding author. Tel.: +81 92 642 2898; fax: +81 92 642 2907. E-mail address: makotoi@agr.kyushu-u.ac.jp (M. Ito). 1 Present address: Division of Glyco-Signal Research, Institute of Molecular Biomembrane and Glycobiology, Tohoku Pharmaceutical University, 4-4-1, Komatsushima, Aoba-ku, Sendai 981-8558, Japan. 1388-1981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2011.02.004 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbalip