title: Identification and characterization of sulfated carbohydrate-binding protein from Lactobacillus reuteri.
abstract: We previously purified a putative sulfated-galactosylceramide (sulfatide)-binding protein with a molecular weight of 47 kDa from the cell surface of Lactobacillus reuteri JCM1081. The aim of this study was to identify the 47-kDa protein, examine its binding to sulfated glycolipids and mucins, and evaluate its role in bacterial adhesion to mucosal surfaces. By cloning and sequencing analysis, the 47-kDa protein was identified as elongation factor-Tu (EF-Tu). Adhesion properties were examined using 6 × Histidine-fused EF-Tu (His6-EF-Tu). Surface plasmon resonance analysis demonstrated pH-dependent binding of His6-EF-Tu to sulfated glycolipids, but not to neutral or sialylated glycolipids, suggesting that a sulfated galactose residue was responsible for EF-Tu binding. Furthermore, His6-EF-Tu was found to bind to porcine gastric mucin (PGM) by enzyme-linked immunosorbent assay. Binding was markedly reduced by sulfatase treatment of PGM and in the presence of acidic and desialylated oligosaccharide fractions containing sulfated carbohydrate residues prepared from PGM, demonstrating that sulfated carbohydrate moieties mediated binding. Histochemical staining revealed similar localization of His6-EF-Tu and high iron diamine staining in porcine mucosa. These results indicated that EF-Tu bound PGM via sulfated carbohydrate moieties. To characterize the contribution of EF-Tu to the interaction between bacterial cells and PGM, we tested whether anti-EF-Tu antibodies could inhibit the interaction. Binding of L. reuteri JCM1081 to PGM was significantly blocked in a concentration-dependent matter, demonstrating the involvement of EF-Tu in bacterial adhesion. In conclusion, the present results demonstrated, for the first time, that EF-Tu bound sulfated carbohydrate moieties of sulfated glycolipids and sulfomucin, thereby promoting adhesion of L. reuteri to mucosal surfaces.
authors.affiliation: Department of Animal Science, Kitasato University School of Veterinary Medicine, Towada, Aomori, Japan.
authors.forename: Keita
authors.initials: K
authors.lastname: Nishiyama
authors.affiliation: Department of Animal Science, Kitasato University School of Veterinary Medicine, Towada, Aomori, Japan.
authors.forename: Ayaka
authors.initials: A
authors.lastname: Ochiai
authors.affiliation: Department of Parasitology, Kitasato University School of Medicine, Sagamihara, Kanagawa, Japan.
authors.forename: Daigo
authors.initials: D
authors.lastname: Tsubokawa
authors.affiliation: Graduate School of Medical Sciences, Kitasato University, Sagamihara, Kanagawa, Japan.
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Ishihara
authors.affiliation: Department of Animal Science, Kitasato University School of Veterinary Medicine, Towada, Aomori, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Yamamoto
authors.affiliation: Department of Animal Science, Kitasato University School of Veterinary Medicine, Towada, Aomori, Japan.
authors.forename: Takao
authors.initials: T
authors.lastname: Mukai
mesh_terms: D000818:Animals; D001422:Bacterial Adhesion / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005753:Gastric Mucosa / Q000378:metabolism; D006017:Glycolipids / Q000378:metabolism*; D052200:Limosilactobacillus reuteri / Q000254:growth & development / Q000378:metabolism*; D009077:Mucins / Q000378:metabolism*; D010444:Peptide Elongation Factor Tu / Q000378:metabolism*; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013552:Swine
chemical_list: D006017:Glycolipids; D009077:Mucins; D011956:Receptors, Cell Surface; C055213:saccharide-binding proteins; C011117:sulfoglycolipids; C012564:sulfomucin; D010444:Peptide Elongation Factor Tu