title: Physicochemically stable cholera toxin B subunit pentamer created by peripheral molecular constraints imposed by de novo-introduced intersubunit disulfide crosslinks.
abstract: We attempted to generate a physicochemically stable cholera toxin B subunit (CTB) by de novo-introduction of intersubunit disulfide bonds between adjacent subunits. Genes encoding double mutant CTB (dmCTB) encompassing a pair of amino acids to be replaced with cysteine residues either at the N-terminal (T1C/T92C, Q3C/T47C), C-terminal (F25C/N103C, Y76C/N103C), or at the internal α-helix region (L77C/T78C), were engineered. One mutant with the N-terminal constraint [dmCTB(T1C/T92C)], expressed as pentamer retained monosialoganglioside G(M1) (GM1) binding affinity, and exhibited robust thermostability. However, when the mutant CTB was heat-treated in the presence of a reducing agent, the thermostable phenotype was abolished, indicating the observed phenotype is due to the introduction of intersubunit disulfide bonds. The mutant CTB also exhibited a strong acid stability at a pH as low as 1.2, as well as stability against incubation with sodium dodecyl sulfate at concentrations as high as 10%. Furthermore, intranasal administration of the mutant CTB to mice induced CTB-specific serum IgG even after heat treatment, while the wildtype CTB failed to show such heat-resistant mucosal immunogenicity. This study demonstrated that an enterotoxin B subunit could be transformed into a physicochemically stable pentamer by the de novo-introduction of peripherally arranged intersubunit disulfide crosslinks, which may prove to be a useful strategy for the development of molecularly stable enterotoxin B subunit-based vaccines and delivery molecules.
authors.affiliation: Molecular Microbiology Group, Department of Tropical Infectious Diseases, COMB, Tropical Biosphere Research Center, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Miyata
authors.forename: Satoshi
authors.initials: S
authors.lastname: Oshiro
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Harakuni
authors.forename: Toki
authors.initials: T
authors.lastname: Taira
authors.forename: Goro
authors.initials: G
authors.lastname: Matsuzaki
authors.forename: Takeshi
authors.initials: T
authors.lastname: Arakawa
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002772:Cholera Toxin / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism*; D005260:Female; D005677:G(M1) Ganglioside / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D018928:Immunity, Mucosal; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D013696:Temperature
chemical_list: D004220:Disulfides; D050505:Mutant Proteins; D011994:Recombinant Proteins; D005677:G(M1) Ganglioside; D002772:Cholera Toxin