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Fucosylation of glycoproteins and glycolipids: opposing roles in cholera intoxication

Nature Chemical Biology volume 21, pages 555–566 (2025)Cite this article

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Abstract

Cholera toxin (CT) is the etiological agent of cholera. Here we report that multiple classes of fucosylated glycoconjugates function in CT binding and intoxication of intestinal epithelial cells. In Colo205 cells, knockout (KO) of B3GNT5, which encodes an enzyme required for synthesis of lacto and neolacto series glycosphingolipids (GSLs), reduces CT binding but sensitizes cells to intoxication. Overexpressing B3GNT5 to generate more fucosylated GSLs confers protection against intoxication, indicating that fucosylated GSLs act as decoy receptors for CT. KO of B3GALT5 causes increased production of fucosylated O-linked and N-linked glycoproteins and leads to increased CT binding and intoxication. KO of B3GNT5 in B3GALT5-KO cells eliminates production of fucosylated GSLs but increases intoxication, identifying fucosylated glycoproteins as functional receptors for CT. These findings provide insight into the molecular determinants regulating CT sensitivity of host cells.

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Fig. 1: CRISPR screen for factors influencing CTB binding to Colo205 cells identifies GSL biosynthetic pathway genes.
Fig. 2: KO of B3GALT5 and B3GNT5 results in increased sensitivity to CT.
Fig. 3: GSLs are decoy receptors for CT.
Fig. 4: B3GALT5-KO cells exhibit increased fucosylation on O-linked glycoproteins.
Fig. 5: CTB binding to Colo205 cells depends on the noncanonical glycan-binding pocket.
Fig. 6: Fucosylated glycoproteins and fucosylated glycolipids have opposing roles in CT intoxication.

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Data availability

Data used to generate Fig. 1b,d are available from the Gene Expression Omnibus under accession number GSE242156. MS data used to generate Fig. 3a, Extended Data Fig. 4a–d, Fig. 4a, Extended Data Fig. 7a–c, Extended Data Fig. 8a–c and Extended Data Fig. 6a are available from GlycoPOST under accession number GPST000467. MS data used to generate Extended Data Fig. 6b,c are available from GlycoPOST under accession number GPST000465. Replicate immunoblot and lectin blot data are available from the Texas Data Repository (https://doi.org/10.18738/T8/N9NF8B). Source data are provided with this paper.

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Acknowledgements

We thank A. Singla, D. Andrade Silva and M. Burns for comments on the manuscript. We thank E. Capota and D. Andrade Silva for technical support. We thank A. Wands, D. Carroll, H. Wu, H. Khan and M. Shiloh for advice and reagents. We thank the UT Southwestern Proteomics Core Facility and its director, A. Lemoff. We acknowledge support from the National Institutes of Health (R01GM090271 and R35GM145599 to J.J.K. and R24GM137782 to P.A.), the Swedish Cancer Foundation (22 2079 Pj to S.T.) and the Welch Foundation (I-1686 to J.J.K.). M.T.G. received support from the NIH (T32GM145467). We thank C. Cairo (University of Alberta) for the RhtrECI plasmid and R. Schnaar (Johns Hopkins) for the P4 inhibitor.

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Authors and Affiliations

  1. Department of Biochemistry, UT Southwestern Medical Center, Dallas, TX, USA

    Atossa C. Ghorashi, Rohit Sai Reddy Konada & Jennifer J. Kohler

  2. Department of Microbiology and Immunology, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden

    Andrew Boucher & Ulf Yrlid

  3. Complex Carbohydrate Research Center, The University of Georgia, Athens, GA, USA

    Stephanie A. Archer-Hartmann, Mehrnoush Taherzadeh Ghahfarrokhi, Nathan B. Murray & Parastoo Azadi

  4. Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden

    Dani Zalem & Susann Teneberg

  5. McDermott Center for Human Growth and Development, UT Southwestern Medical Center, Dallas, TX, USA

    Xunzhi Zhang & Chao Xing

  6. Department of Bioinformatics, UT Southwestern Medical Center, Dallas, TX, USA

    Chao Xing

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  1. Atossa C. Ghorashi
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A.C.G. and J.J.K. conceptualized the project and experimental approach, with input from A.B., R.S.R.K. and U.Y. A.C.G. conducted all cell culture experiments, flow cytometry experiments, functional assays and immunoblot experiments. A.B. prepared biotinylated CTB and CTB mutants with supervision from U.Y. S.A.A.-H. conducted MS analysis of intact glycolipids with supervision from P.A. M.T.G. performed MS analysis of procainamide-labeled glycans from glycolipids with supervision from P.A. D.Z. isolated neutral glycolipids and conducted MS analysis of their glycans with supervision from S.T. N.B.M. and S.A.A.-H. conducted N-linked and O-linked glycomic analyses with supervision from P.A. R.S.R.K. prepared EGCase and conducted preliminary analyses of glycolipids. X.Z. performed bioinformatics analyses with supervision from C.X. A.C.G. and J.J.K. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Jennifer J. Kohler.

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Nature Chemical Biology thanks the anonymous reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Characterization of B3GALT5-KO and B3GALT5-KO + OE cells.

(A, B) Representative histograms (left panel) from the flow cytometry analyses of cell surface binding of Lewis a antibody (A) or Lewis x antibody (B) to indicated cell lines. Bar graphs (right panel) show quantification from 3 independent trials. Error bars indicate mean ± SD. (C) Quantification of gMFI from flow cytometry analyses of cells treated with increasing concentrations of CTB. Data shown are from 3 independent trials and normalized to the maximum APC signal in WT cells. Error bars indicate mean ± SD. Indicated cell lines were incubated for 72 h with increasing concentrations of CTB-Saporin (D) or unconjugated saporin (E). Cell survival upon internalization of CTB-saporin was measured using the Cell Titer-Glo 2.0 assay. Data shown are luminescence values normalized to the signal from the untreated condition for each cell type. Each datapoint is a biological replicate consisting of 3 averaged technical replicates. Error bars indicate mean ± SD of 3 biological replicates. (F) Cells pretreated with BFA or vehicle control for 0.5 h were incubated for 1.5 h with CT (1 nM) or buffer alone. Accumulation of cAMP was measured. Data shown are inverse of luminescence values normalized first to the total amount of cells plated for each cell line, then to the signal in CT-treated control cells. Each datapoint is a biological replicate consisting of 3 averaged technical replicates. Error bars indicate mean ± SD of 3 biological replicates. (G) Control, B3GALT5-KO m1, m2 and KO + OE cells were treated with forskolin (10 µM) for 0.5 h and then analyzed as in panel F. Statistical analyses for panels A and B were performed by one-way ANOVA with Tukey correction and for panels C, F, and G by two-way ANOVA with Tukey correction.‘ns’ indicates not significant, **** indicates adjusted P-value < 0.0001. Exact P-values are as follows: 0.0004 and 0.0412 for control versus B3GALT5-KO m2 and B3GALT5-KO + OE; 0.0041 and 0.0024 for B3GALT5-KO m1 versus B3GALT5-KO m2 and B3GALT5-KO + OE, respectively (panel B); 0.0390 for control versus B3GALT5-KO m2 treated with 0.0375 μg/mL; 0.0002 and 0.0014 for control versus B3GALT5-KO m2 or m1 treated with 0.075 μg/mL, respectively (panel C).

Source data

Extended Data Fig. 2 Characterization of B3GNT5-KO and B3GNT5-KO + OE cells.

(A) Quantification of gMFI from flow cytometry analyses of cells treated with increasing concentrations of CTB. Data shown are from 3 independent trials and normalized to the maximum APC signal in WT cells. Error bars indicate mean ± SD. (B, C) Representative histograms (left panel) from the flow cytometry analyses of cell surface binding of Lewis x antibody (B) or Lewis a antibody (C) to control, B3GNT5-KO m1, m2 and KO + OE cells. Bar graphs (right panel) show quantification from 3 independent trials. Control, B3GNT5-KO m1 and KO + OE cells were incubated for 72 h with increasing concentrations of CTB-Saporin (D) or unconjugated saporin (E). Cell survival upon internalization of CTB-saporin was measured using the Cell Titer-Glo 2.0 assay. Data shown are luminescence values normalized to the signal from the untreated condition for each cell type. Each datapoint is a biological replicate consisting of 3 averaged technical replicates. Error bars indicate mean ± SD of 3 biological replicates. (F) Cells pretreated with BFA or a vehicle control for 0.5 h were incubated for 1.5 h with CT (1 nM) or buffer alone. Accumulation of cAMP was measured. Data shown are inverse of luminescence values normalized first to the total amount of cells plated for each cell line, then to the signal in CT-treated control cells. Each datapoint is a biological replicate consisting of 3 averaged technical replicates. Error bars indicate mean ± SD of 3 biological replicates. (G) Control, B3GNT5-KO m1, m2 and KO + OE cells were treated with forskolin (10 µM) for 0.5 h and then analyzed as in panel F. Statistical analyses for panels B and C were performed by one-way ANOVA with Tukey correction and for panels A, D, E, F, and G by two-way ANOVA with Tukey correction. ‘ns’ indicates not significant, **** indicates adjusted P-value < 0.0001. Exact P-values are as follows: 0.0219 for control versus B3GNT5-KO m1 treated with 1.25 μg/mL; 0.0010 and 0.0077 for control versus B3GNT5-KO m1 or m2 treated with 2.5 μg/mL, respectively (panel A); 0.0059 and 0.0036 for control versus B3GNT5-KO + OE treated with 2.5 or 12.5 μg/mL CTB-saporin (panel D).

Source data

Extended Data Fig. 3 Characterization of B3GALT5 + B3GNT5-dKO cells.

(A) Representative histograms (left panel) from the flow cytometry analyses of cell surface binding of Lewis x antibody to control, B3GALT5 -KO m1, B3GNT5-KO m1, and B3GALT5 + B3GNT5-dKO cells. Bar graph (right panel) shows quantification from 3 independent trials. (B, C) Control, B3GALT5-KO m1, and B3GALT5 + B3GNT5-dKO cells were incubated for 72 h with increasing concentrations of CTB-Saporin (B) or unconjugated saporin (C). Cell survival upon internalization of CTB-saporin measured using the Cell Titer-Glo 2.0 assay. Data shown are luminescence values normalized to the signal from the untreated condition for each cell type. Each datapoint is a biological replicate consisting of 3 averaged technical replicates. Error bars indicate mean ± SD of 3 biological replicates. (D) Cells pretreated with BFA or a vehicle control for 0.5 h were incubated for 1.5 h with CT (1 nM) or buffer alone. Accumulation of cAMP was measured. Data shown are inverse of luminescence values normalized first to the total amount of cells plated for each cell line, then to the signal in CT-treated control cells. Each datapoint is a biological replicate (n = 2) consisting of 3 averaged technical replicates. (E) Control, B3GALT5-KO m1, m2 and KO + OE cells were treated with forskolin (10 µM) for 0.5 h and then analyzed as in panel D. Each datapoint is a biological replicate consisting of 3 averaged technical replicates. Error bars indicate mean ± SD of 3 biological replicates. Statistical analyses for panel A were performed by one-way ANOVA with Tukey correction and for panels B, C, D, and E by two-way ANOVA with Tukey correction. **** indicates adjusted P-value < 0.0001. Exact P-values are as follows: 0.0003 and 0.0048 for B3GALT5 + B3GNT5-dKO versus control or B3GALT5-KO m1, respectively (panel A); 0.0002 and 0.0171 for control versus B3GALT5 + B3GNT5-dKO treated with 2.5 μg/mL CTB saporin or 12.5 μg/mL saporin (panels C and D, respectively).

Source data

Extended Data Fig. 4 Fucosylated lacto-series GSLs detected in control but not KO cell lines.

MS analysis of GSLs from control (A), B3GALT5-KO m1 (B), and B3GNT5-KO m1 (C) cells. (D) Example MS/MS spectrum of a fucosylated lacto-series glycolipid detected in control cells, confirming structure.

Extended Data Fig. 5 Validation of GSLs as decoy receptors for CT.

(A) Lectin blot with CTB-biotin of control and B3GALT5-KO m1 cell lysates, and pure GM1. Samples were treated for 16 h with endoglycoceramidase or a vehicle control. Data shown are a single representative trial of 3 independent biological replicates. (B-G) Control, B3GNT5-KO + OE, and B3GALT5-KO m1 cells were treated with P4 inhibitor of glycosphingolipid biosynthesis for 72 h then lysed for lectin blot analysis (B) or treated with BFA or a vehicle control for 0.5 h prior to incubation with CT (1 nM) for analysis of cAMP accumulation (C, D, and E). Alternately, cells were treated with forskolin (10 µM) for 0.5 h (F and G). Accumulation of cAMP was measured. Data shown are inverse of luminescence values normalized first to the total amount of cells plated for each cell line, then to the signal in CT-treated control cells. Each datapoint is a biological replicate (n = 2 in panel E, n = 3 in panels C, D, F, and G) consisting of 3 averaged technical replicates. Error bars indicate mean ± SD of 3 biological replicates. Statistical analyses were performed by two-way ANOVA with Tukey correction. Exact P-values are 0.0002 for control versus inhibitor-treated B3GALT5-KO m1 as well as inhibitor-treated control versus untreated B3GALT5-KO m1; for inhibitor-treated control versus inhibitor-treated B3GALT5-KO m1, the exact P-value is 0.0052 (panel C).

Source data

Extended Data Fig. 6 Analysis of GSLs from B3GNT5-KO + OE cells.

(A) Glycolipids were isolated from B3GALT5-KO m1 or B3GNT5-KO + OE cells. Glycans were released with endoglycoceramidase, labeled with procainamide, and analyzed by mass spectrometry with HILIC-FL separation. (B, C) LC-ESI/MS analysis of oligosaccharides obtained by digestion of neutral GSLs from B3GNT5-KO + OE cells with endoglycoceramidase I. Diagnostic ions indicate carbohydrate linkage positions. (C) An MS spectrum displaying a series of C type fragment ions (C2α at m/z 528, C3α at m/z 690, C4α at m/z 1039, C5α at m/z 1201, C6 at m/z 1550, and C7 at m/z 1712), which identified an oligosaccharide with Hex-(Fuc-)HexNAc-Hex-(Fuc-)HexNAc-Hex-(Fuc-)HexNAc-Hex-Hex sequence. The ion at m/z 364 is obtained by double glycosidic cleavage of the 3-linked branch (C2/Z3β), and characteristic for an internal 4-linked GlcNAc substituted with a Fuc at 3-position that is a terminal Lex (refs. 90,94). Taken together this indicated an undecasaccharide with a terminal Lex determinant. (D, E) B3GALT5 + B3GNT5-dKO cells were incubated with GSLs extracted from B3GNT5-KO + OE cells, a commercial mixture of neutral GSLs, or purified GM1. Cells were then treated with BFA or a vehicle control for 0.5 h prior to incubation with CT (1 nM) for analysis of cAMP accumulation (D). Alternately, cells were treated with forskolin (10 µM) for 0.5 h (n = 2) (E). Accumulation of cAMP was measured. Data shown are inverse of luminescence values normalized first to the total amount of cells plated for each cell line, then to the signal in CT-treated control cells. Each datapoint is a biological replicate (n = 3 for panel D, n = 2 for panel E) consisting of 2 averaged technical replicates. Error bars indicate mean ± SD of 3 biological replicates (panel D). Statistical analyses were performed by two-way ANOVA with Tukey correction. No significant differences were observed.

Source data

Extended Data Fig. 7 B3GALT5-KO cells exhibit increased fucosylation on N-linked glycoproteins.

(A) N-linked glycoforms detected in control and B3GALT5-KO m1 cells by LC-MS/MS analysis. Quantification is based on triplicate analysis. (B) Example MS/MS of a complex, fucosylated N-linked glycan detected in B3GALT5-KO m1 cells confirming placement of fucose. (C) Bar graphs showing the relative enrichment of mono- vs di- vs tri-fucosylated N-linked glycans detected by LC-MS/MS analysis of control and B3GALT5-KO m1 cells. Statistical analysis was performed by two-tailed t-test with Holm-Šídák correction. Exact adjusted P-value = 0.013508 for mono-fucosylated N-linked glycans detected in control versus B3GALT5-KO m1. Exact adjusted P-value = 0.014105 for both di- and tri-fucosylated N-linked glycans detected in B3GALT5-KO m1 versus control.

Extended Data Fig. 8 B3GALT5-KO cells exhibit increased fucosylation on O-linked glycoproteins.

(A) O-linked glycoforms detected in control and B3GALT5-KO m1 cells by LC-MS/MS analysis. Quantification is based on triplicate analysis. (B) Example MS/MS of a fucosylated O-linked glycan detected in B3GALT5-KO m1 cells. (C) Example MS/MS of core 2 O-linked glycan detected in B3GALT5-KO m1 cells confirming structure. (D, E) B3GALT5-KO m1 cells were rescued with either WT or catalytically dead (mut) B3GALT5-OE. Rescued cells were treated with BFA or a vehicle control for 0.5 h prior to incubation with CT (1 nM) for analysis of cAMP accumulation (D). Alternately, cells were treated with forskolin (10 µM) for 0.5 h (E). Accumulation of cAMP was measured. Data shown are inverse of luminescence values normalized first to the total amount of cells plated for each cell line, then to the signal in CT-treated control cells. Each datapoint is a biological replicate consisting of 2 averaged technical replicates. Error bars indicate mean ± SD of 3 biological replicates. Statistical analyses were performed by two-way ANOVA with Tukey correction. No significant differences were observed.

Source data

Extended Data Fig. 9 Validation of SLC35C1-KO cell lines.

(A) GM1 was treated with EGCase or vehicle, then detected by lectin blot using WT CTB-biotin, W88K CTB-biotin, and H18L CTB-biotin. Data presented are from 3 distinct membranes processed simultaneously (separated by vertical lines) and are a single representative trial from 3 biological replicates. (B, C) Representative histograms (left panel) from flow cytometry analyses of anti-Lex antibody (B) and AAL (C) binding to surfaces of control, B3GALT5-KO m1, SLC35C1-KO, and B3GALT5 + SLC35C1-dKO cells. Quantification of gMFIs (right panel) from 3 biological replicates are normalized to the maximum signal in control cells. Lysates from control, B3GALT5-KO m1, SLC35C1-KO and B3GALT5 + SLC35C1-dKO cells were analyzed by immunoblot probing with anti-Lex antibody or lectin blot probing with AAL (D). Data shown are a single representative trial from 3 biological replicates. (E) Control, B3GALT5-KO m1, SLC35C1-KO and B3GALT5 + SLC35C1-dKO cells were incubated for 72 h with unconjugated saporin (12.5 μg/mL). Survival data shown are luminescence values normalized to the signal from the untreated condition for each cell type. Each datapoint indicates the mean of 2 biological replicates, each consisting of 3 averaged technical replicates. (F) Cells pretreated with BFA or a vehicle control for 0.5 h were incubated for 1.5 h with CT (1 nM) or buffer alone. Accumulation of cAMP was measured. Data shown are inverse of luminescence values normalized first to the total amount of cells plated for each cell line, then to the signal in CT-treated control cells. Each datapoint is a biological replicate (n = 2) consisting of 3 averaged technical replicates. (G) Cells were treated with forskolin (10 µM) for 0.5 h and then analyzed as in panel F. Each datapoint is a biological replicate consisting of 3 averaged technical replicates. Error bars indicate mean ± SD of 3 biological replicates. Statistical analyses were performed by one-way (panels B and C) or two-way (panels F and G) ANOVA with Tukey correction. **** indicates adjusted P-value < 0.0001. Exact P-value for control versus B3GALT5-KO m1 treated with AAL is 0.0018.

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Extended Data Fig. 10 CTB-interacting glycoproteins.

(A) Top 50 proteins identified from CTB-biotin pulldown from B3GALT5 + B3GNT5-dKO versus B3GALT5 + SLC35C1-dKO cell lysates. Fold-change is the abundance in the B3GALT5 +  B3GNT5-dKO pulldown as compared to the B3GALT5 + SLC35C1-dKO pulldown. Unadjusted P-values are derived from two-tailed student’s t-test. (B) Gene ontology (GO) analysis using Fisher’s exact test with Bonferroni correction to identify pathways enriched in CTB-biotin pulldown from B3GALT5 + B3GNT5-dKO cell lysates as compared to B3GALT5 + SLC35C1-dKO cell lysates. Top 25 pathways are shown. (C) Two hits (FLOT1 and MUC1, boldface font in panel A) were selected for confirmation. Total lysates and material from CTB-biotin pulldowns were analyzed by immunoblot using antibodies against FLOT1 and MUC1.

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Supplementary information

Supplementary Information

Supplementary Fig. 1.

Reporting Summary

Supplementary Table 1

Reagents list.

Supplementary Table 2

Oligonucleotide list.

Supplementary Table 3

CRISPR KO sequence validation.

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Ghorashi, A.C., Boucher, A., Archer-Hartmann, S.A. et al. Fucosylation of glycoproteins and glycolipids: opposing roles in cholera intoxication. Nat Chem Biol 21, 555–566 (2025). https://doi.org/10.1038/s41589-024-01748-5

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