Structure of Rhamnose-binding Lectin CSL3: Unique Pseudo-tetrameric Architecture of a Pattern Recognition Protein Tsuyoshi Shirai 1,2 ⁎, Yasuharu Watanabe 3 , Min-sub Lee 3 , Tomohisa Ogawa 2,3 and Koji Muramoto 3 1 Department of Bioscience, Nagahama Institute of Bio-science and Technology, and JST-BIRD, 1266 Tamura, Nagahama 526-0829, Japan 2 Center for Interdisciplinary Research, Tohoku University, Aramaki, Aoba-ku, Sendai 980-8578, Japan 3 Graduate School of Life Sciences, Tohoku University, 1-1Tsutsumidori-amamiyamachi, Aoba-ku, Sendai 981-8555, Japan Received 20 April 2009; received in revised form 4 June 2009; accepted 8 June 2009 Available online 12 June 2009 The crystal structure of the L-rhamnose-binding lectin CSL3 was determined to 1.8 Å resolution. This protein is a component of the germline-encoded pattern recognition proteins in innate immunity. CSL3 is a homodimer of two 20 kDa subunits with a dumbbell-like shape overall, in which the N- and C-terminal domains of different subunits form lobe structures connected with flexible linker peptides. The complex structures of the protein with specific carbohydrates demonstrated the importance of the most variable loop region among homologues for the specificity toward oligosaccharides. CSL3 and Shiga-like toxin both use Gb 3 as a cellular receptor to evoke apoptosis. They have very different overall architecture but share the separation distance between carbohydrate-binding sites. An inspection of the structure database suggested that the pseudo-tetrameric structure of CSL3 was unique among the known lectins. This architecture implies this protein might provide a unique tool for further investigations into the relationships between architecture and function of pattern recognition proteins. © 2009 Elsevier Ltd. All rights reserved. Edited by Dr K. Morikawa Keywords: lectin; innate immunity; globotriose; X-ray crystallography; bioinformatics Introduction The L-rhamnose-binding lectins (RBLs) are a family of animal lectins that bind specifically to L-rhamnose or D-galactose. 1,2 RBLs have been found in over 25 species of fish, 3–8 sea urchin, 9 oyster, 10 and ascidian. 11 RBLs are thought to be an important component of germline-encoded pattern recognition proteins (PRP) in innate immunity. 12–14 They are localized in tissues of gills, intestine, and spleen, which are related to the immune system. 8,15 RBLs agglutinate Gram-negative and Gram-positive bacteria by recognizing the cell- surface lipopolysaccharides and lipoteichoic acid, respectively. 16,17 RBLs bind to glycolipids and glyco- proteins of the microsporidian fish pathogens, 8,18 and the RBL receptor was expressed on peritoneal macro- phages of fishes after an inflammatory stimulation. 19,20 Three RBLs, CSL1, 2, and 3, from eggs of the chum salmon (Oncorhynchus keta) were found to induce proinflammatory cytokines, including IL-1b1, IL-1b2, TNF-α1, TNF-α2, and IL-8, by recognizing Gb 3 (globotriose ceramide; Galα1-4Galβ1-4Galβ1-Cer, also known as CD77) on the surface of a peritoneal macrophage (RTM5) and an established fibroblastic- like (RTG-2) cell line of rainbow trout. 8 CSLs were shown to have an opsonic effect on RTM5 by binding to Gb 3 . 14 Gb 3 , which is located in lipid rafts and is up-regulated through immune responses, 21–23 is known also as the functional receptor for pathogenic bacterial toxins, such as Shiga toxin from Shigella dysenteriae serotype 1, Shigella diarrhea , and ⁎Corresponding author. E-mail address: t_shirai@nagahama-i-bio.ac.jp. Abbreviations used: RBL, L-rhamnose-binding lectin; PRP, pattern recognition protein; SUEL, sea urchin egg lectin; PPMP, DL-threo-1-phenyl-2-palmitoylamino-3- morpholino-1-propanol; SLT, Shiga-like toxin; FITC, fluorescein isothiocyanate. doi:10.1016/j.jmb.2009.06.027 J. Mol. Biol. (2009) 391, 390–403 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2009 Elsevier Ltd. All rights reserved.