Physicochemically stable cholera toxin B subunit pentamer created by peripheral molecular constraints imposed by de novo-introduced intersubunit disulfide crosslinks

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• 作者：Takeshi Miyata^a ; ¹ ; Satoshi Oshiro^b ; ¹ ; Tetsuya Harakuni^a ; ¹ ; Toki Taira^b ; Goro Matsuzaki^a ; ^c ; Takeshi Arakawa^a ; ^c ; ¹ ; ^{tarakawa@comb.u-ryukyu.ac.jp}
• 关键词：Vaccine ; Adjuvant ; AB5 enterotoxin ; Cholera toxin B subunit pentamer ; De novo intersubunit disulfide bonds ; Physicochemically stable
• 刊名：Vaccine
• 出版年：2012
• 期刊代码：89_0264410x
• 类别：imm
• 出版时间：13 June, 2012
• 卷：30
• 期：28
• 页码：4225-4232
• 文件大小：1223 K

摘要

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 伪-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.