Journal: Nature Communications

Article Title: Gut mucin fucosylation dictates the entry of botulinum toxin complexes

doi: 10.1038/s41467-025-65384-w

Figure Lengend Snippet: a Survival curves of BALB/c mice ( n = 5 per group) administered large progenitor toxin complexes (L-PTC/A 62A or L-PTC/B Okra ) intraperitoneally (i.p.; 50 pg) or intragastrically (i.g.; 50 or 1000 ng). b , c Representative images of whole-mounted small intestine and L-PTCs. A mixture of Alexa Fluor (AF) 568–labeled L-PTC/A 62A (red) and AF 488–labeled L-PTC/B Okra (green) was injected into ligated mouse intestinal loops. M cells ( b ) and mucin ( c ) were visualized with anti-glycoprotein 2 (GP2) and anti-MUC2 antibodies, respectively (blue). VE villous epithelium, FAE follicle-associated epithelium. Scale bars, 50 µm (low magnification) in upper panels of b , g ; 20 µm (high magnification) in lower panels of b , c . d Mucin penetration of L-PTCs was quantified using transwell inserts coated with porcine gastric mucin (PGM). Upper-to-lower penetration was analyzed by immunoblotting for L-PTCs and normalized to the bovine serum albumin (BSA) control. e Schematic representation of a mucus-depleted mouse model. Mice were gavaged with vehicle (sterile water) or 100 mg/mL N -acetylcysteine (NAC) 1 h before the in situ loop assay and challenge. f Small intestinal mucins were collected from vehicle- or NAC-treated mice. To quantify the mucin concentration, binding of wheat germ agglutinin (WGA) to the mucin was analyzed by ELISA. g Binding of L-PTCs (green) to the villous epithelium. Nuclei and mucin were visualized with Hoechst 33342 (blue) and an anti-MUC2 antibody (red). Arrows indicate L-PTC attachment to the epithelium. h NAC-treated mice ( n = 5 per group) were challenged with L-PTC/A 62A (1000 ng) and L-PTC/B Okra (50 ng). d , f Values represent the mean ± SD of triplicate ( d ) and four independent samples ( f ), two-tailed Student’s t- test. a , h Log-rank test.

Article Snippet: PGM (Merck, M1778), MIM, and human intestinal mucin were separately coated on 96-well ELISA plates (Iwaki).

Techniques: Labeling, Injection, Western Blot, Control, Sterility, In Situ, Concentration Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Gut mucin fucosylation dictates the entry of botulinum toxin complexes

doi: 10.1038/s41467-025-65384-w

Figure Lengend Snippet: a, b Binding of PTCs (L-PTC, BoNT+NTNHA + HA; M-PTC, BoNT+NTNHA) ( a ) or HAs ( b ) to porcine gastric mucin (PGM) was analyzed by ELISA. c Representative images of whole-mounted small intestine with AF 568–labeled HA/A 62A (red) and AF 488–labeled HA/B Okra (green). M cells were visualized with anti-GP2 antibody (blue). Scale bars, 50 µm (upper panels) and 20 µm (lower panels). d Chimeric recombinant L-PTCs (rL-PTCs) were reconstituted from BoNT/B Okra , NTNHA/B Okra , and HA (BB for HA/B Okra , BA for HA/A 62A ). Purified proteins were verified by SDS-PAGE with Coomassie blue staining. e Mice were challenged with the rL-PTC/BA (red) or rL-PTC/BB (blue) via intraperitoneal injection (i.p., 100 pg; n = 10 per group) or intragastric administration (i.g., 200 ng; n = 35 for rL-PTC/BA and n = 28 for rL-PTC/BB). f Carbohydrate-binding sites of HA (HA1/A 62A -Lac: PDB ID 4LO2 ; HA1/B Okra -Lac: 4OUJ ; HA3/A 62A -6SL: 4LO5 ; HA3/B Okra -6SL: 9UG6, this work). Ligands and interacting amino acids are shown in stick models colored light red (HA/A 62A ), light blue (HA/B Okra ), yellow (galactose, Gal), blue (glucose, Glc), and purple (Neu5Ac). The omit map of the HA3/B Okra -6SL structure is shown in Supplementary Fig. . g ELISA was used to analyze the binding of mutated HAs to PGM. N285/6A HA1 and R528A HA3 are galactose- and sialic acid-binding–defective mutants, respectively . NRA, N285/6A HA1 /R528A HA3 . HNRK, H281N HA1 /R619K HA3 . NHKR, N282H HA1 /K619R HA3 . h Ligated mouse intestinal loop assay with mutated HAs (red). Nuclei and mucin were visualized with Hoechst 33342 (blue) and an anti-MUC2 antibody (green). Arrows indicate HA attachment to the epithelium. Scale bars, 50 µm. a , b , g Values represent the mean ± SD of triplicate wells.

Article Snippet: PGM (Merck, M1778), MIM, and human intestinal mucin were separately coated on 96-well ELISA plates (Iwaki).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Labeling, Recombinant, Purification, SDS Page, Staining, Injection