title: Bacillus cereus sphingomyelinase recognizes ganglioside GM3.
abstract: Sphingomyelinase (SMase) from Bacillus cereus (Bc-SMase) hydrolyzes sphingomyelin (SM) to phosphocholine and ceramide in a divalent metal ion-dependent manner, and is a virulence factor for septicemia. Bc-SMase has three characteristic sites, viz., the central site (catalytic site), side-edge site (membrane binding site), and β-hairpin region (membrane binding site). Here, we show that the β-hairpin directly binds to gangliosides, especially NeuAcα2-3Galβ1-4Glcβ1-1ceramide (GM3) through a carbohydrate moiety. Neuraminidase inhibited the binding of Bc-SMase to mouse peritoneal macrophages in a dose-dependent manner. SPR analysis revealed that the binding response of Bc-SMase to liposomes containing GM3 was about 15-fold higher than that to liposomes lacking GM3. Moreover, experiments with site-directed mutants indicated that Trp-284 and Phe-285 in the β-hairpin play an important role in the interaction with GM3. The binding of W284A and F285A mutant enzymes to mouse macrophages decreased markedly in comparison to the binding by wild-type enzymes. Therefore, we conclude that GM3 is the primary cellular receptor for Bc-SMase, and that the β-hairpin region is the tethering region for gangliosides.
authors: Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan. masa@ph.bunri-u.ac.jp
authors: Masataka
authors: M
authors: Oda
authors: Aoi
authors: A
authors: Fujita
authors: Kensuke
authors: K
authors: Okui
authors: Kazuaki
authors: K
authors: Miyamoto
authors: Masahiro
authors: M
authors: Shibutani
authors: Teruhisa
authors: T
authors: Takagishi
authors: Masahiro
authors: M
authors: Nagahama
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001409:Bacillus cereus / Q000201:enzymology*; D005679:G(M3) Ganglioside / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry; D008264:Macrophages / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D013108:Sphingomyelin Phosphodiesterase / Q000737:chemistry* / Q000235:genetics; D020349:Surface Plasmon Resonance
chemical_list: D005679:G(M3) Ganglioside; D008081:Liposomes; D013108:Sphingomyelin Phosphodiesterase