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Cell type and receptor identity regulate cholera toxin subunit B (CTB) internalization

Sethi, A;Wands, A;Mettlen, M;Krishnamurthy, S;Wu, H;Kohler, J;
Product: Cholera Toxin (AZIDE-FREE) from Vibrio cholerae

Cholera toxin (CT) is a secreted bacterial toxin that binds to glycoconjugate receptors on the surface of mammalian cells, enters mammalian cells through endocytic mechanisms and intoxicates mammalian cells by activating cytosolic adenylate cyclase. CT recognizes cell surface receptors through its B subunit (CTB). While the ganglioside GM1 has been historically described as the sole receptor, CTB is also capable of binding to fucosylated glycoconjugates, and fucosylated molecules have been shown to play a functional role in host cell intoxication by CT. Here, we use colonic epithelial and respiratory epithelial cell lines to examine how two types of CT receptors—gangliosides and fucosylated glycoconjugates—contribute to CTB internalization. We show that fucosylated glycoconjugates contribute to CTB binding to and internalization into host cells, even when the ganglioside GM1 is present. The contributions of the two classes of receptors to CTB internalization depend on cell type. Additionally, in a cell line that harbours both classes of receptors, gangliosides dictate the efficiency of CTB internalization. Together, the results lend support to the idea that fucosylated glycoconjugates play a functional role in CTB internalization, and suggest that CT internalization depends on both receptor identity and cell type.

349834982019-03-152019-03-1511:25:1911:25:192019-03-212019-03-2111:20:4611:20:46Sethi, A;Wands, A;Mettlen, M;Krishnamurthy, S;Wu, H;Kohler, J;Sethi, A;Wands, A;Mettlen, M;Krishnamurthy, S;Wu, H;Kohler, J;20192019Cell type and receptor identity regulate cholera toxin subunit B (CTB) internalizationCell type and receptor identity regulate cholera toxin subunit B (CTB) internalizationInterface FocusInterface Focus20180076201800769922

Cholera toxin (CT) is a secreted bacterial toxin that binds to glycoconjugate receptors on the surface of mammalian cells, enters mammalian cells through endocytic mechanisms and intoxicates mammalian cells by activating cytosolic adenylate cyclase. CT recognizes cell surface receptors through its B subunit (CTB). While the ganglioside GM1 has been historically described as the sole receptor, CTB is also capable of binding to fucosylated glycoconjugates, and fucosylated molecules have been shown to play a functional role in host cell intoxication by CT. Here, we use colonic epithelial and respiratory epithelial cell lines to examine how two types of CT receptors—gangliosides and fucosylated glycoconjugates—contribute to CTB internalization. We show that fucosylated glycoconjugates contribute to CTB binding to and internalization into host cells, even when the ganglioside GM1 is present. The contributions of the two classes of receptors to CTB internalization depend on cell type. Additionally, in a cell line that harbours both classes of receptors, gangliosides dictate the efficiency of CTB internalization. Together, the results lend support to the idea that fucosylated glycoconjugates play a functional role in CTB internalization, and suggest that CT internalization depends on both receptor identity and cell type.

Cholera toxin (CT) is a secreted bacterial toxin that binds to glycoconjugate receptors on the surface of mammalian cells, enters mammalian cells through endocytic mechanisms and intoxicates mammalian cells by activating cytosolic adenylate cyclase. CT recognizes cell surface receptors through its B subunit (CTB). While the ganglioside GM1 has been historically described as the sole receptor, CTB is also capable of binding to fucosylated glycoconjugates, and fucosylated molecules have been shown to play a functional role in host cell intoxication by CT. Here, we use colonic epithelial and respiratory epithelial cell lines to examine how two types of CT receptors—gangliosides and fucosylated glycoconjugates—contribute to CTB internalization. We show that fucosylated glycoconjugates contribute to CTB binding to and internalization into host cells, even when the ganglioside GM1 is present. The contributions of the two classes of receptors to CTB internalization depend on cell type. Additionally, in a cell line that harbours both classes of receptors, gangliosides dictate the efficiency of CTB internalization. Together, the results lend support to the idea that fucosylated glycoconjugates play a functional role in CTB internalization, and suggest that CT internalization depends on both receptor identity and cell type.

2.72.7

General chemicals:

... CT (azide-free) from V. cholerae used for cAMP experiments was purchased from List Biological Laboratories (Campbell, CA, USA) (catalogue no. 100B).

cAMP measurement:

One hundred microlitres of 50 000 cells ml−1 cell suspensions of individual cell lines were added in wells in white-walled 96-well plates (Thermo-Fisher Scientific, catalogue no. 07-200-628) and cultured for 72 h. The wells were washed twice with PBS and then treated with 0.1 nM CT holotoxin at 37°C for 60 min (List Biologicals) in complete induction buffer provided in the cAMP-Glo kit (Promega, catalogue no. V1501). The assay was performed following the manufacturer's protocol (cAMP-Glo assay; Promega). The luminescence values were obtained using a Synergy Neo microplate reader (BioTek, Winooski, VT, USA).

General chemicals:

... CT (azide-free) from V. cholerae used for cAMP experiments was purchased from List Biological Laboratories (Campbell, CA, USA) (catalogue no. 100B).

cAMP measurement:

One hundred microlitres of 50 000 cells ml−1 cell suspensions of individual cell lines were added in wells in white-walled 96-well plates (Thermo-Fisher Scientific, catalogue no. 07-200-628) and cultured for 72 h. The wells were washed twice with PBS and then treated with 0.1 nM CT holotoxin at 37°C for 60 min (List Biologicals) in complete induction buffer provided in the cAMP-Glo kit (Promega, catalogue no. V1501). The assay was performed following the manufacturer's protocol (cAMP-Glo assay; Promega). The luminescence values were obtained using a Synergy Neo microplate reader (BioTek, Winooski, VT, USA).

https://royalsocietypublishing.org/doi/abs/10.1098/rsfs.2018.0076https://royalsocietypublishing.org/doi/abs/10.1098/rsfs.2018.00762019-04-062019-04-0610.1098/rsfs.2018.007610.1098/rsfs.2018.0076Cholera Toxin (AZIDE-FREE) from Vibrio choleraeCholera Toxin (AZIDE-FREE) from Vibrio choleraejennifer.kohler@utsouthwestern.edujennifer.kohler@utsouthwestern.eduAdenylate Cyclase;B subunit;Binding;Biological;cAMP;Cell;Cholera;Cholerae;CTB;Cyclase;Endocytic;Epithelial;Ganglioside;GM1;Host;Intoxication;List;List Biological;Mammalian;Receptor;Regulate;Respiratory;Subunit;Surface;Toxin;V. cholerae;Interface FocusAdenylate Cyclase;B subunit;Binding;Biological;cAMP;Cell;Cholera;Cholerae;CTB;Cyclase;Endocytic;Epithelial;Ganglioside;GM1;Host;Intoxication;List;List Biological;Mammalian;Receptor;Regulate;Respiratory;Subunit;Surface;Toxin;V. cholerae;Interface Focus100B100Bcell-type-and-receptor-identitycell-type-and-receptor-identity