Botulinum neurotoxin A complex recognizes host carbohydrates through its hemagglutinin component

Abstract

Botulinum neurotoxins (BoNTs) are potent bacterial toxins. The high oral toxicity of BoNTs is largely attributed to the progenitor toxin complex (PTC), which is assembled from BoNT and nontoxic neurotoxin-associated proteins (NAPs) that are produced together with BoNT in bacteria. Here, we performed ex vivo studies to examine binding of the highly homogeneous recombinant NAPs to mouse small intestine. We also carried out the first comprehensive glycan array screening with the hemagglutinin (HA) component of NAPs. Our data confirmed that intestinal binding of the PTC is partly mediated by the HA moiety through multivalent interactions between HA and host carbohydrates. The specific HA-carbohydrate recognition could be inhibited by receptor-mimicking saccharides.

Figure 1

Architecture of the large progenitor toxin complex (L-PTC) (16S complex). (A) Surface representation of the L-PTC of botulinum neurotoxin (BoNT/A) [17]. The M-PTC (12S complex) is composed of BoNT/A (magenta) and NTNHA (gray). The three-blade shaped hemagglutinin (HA) complex comprises three HA70 (HA3, yellow), three HA17 (HA2, cyan), and six HA33 (HA1, orange). (B) A SDS-PAGE showing the purity of the various recombinant PTC components. Please note that a small amount of HA70 was spontaneously nicked into two peptides (HA70a and HA70b) as previously reported [17]; HA70 and HA17 in the HA-wt complex had uncleaved His-tags. (C) A Western blotting using the anti-myc antibody confirmed the integrity of the myc-tag on HA70 (lanes 1, 3), HA70^D3 (lane 2), and NTNHA (lanes 4, 5).

Figure 2

Intestinal binding of the PTC is primarily mediated by the HA complex. Same amount of the myc-tagged HA-wt, HA-mini, HA-DAFA, M-PTC, and non-toxic non-hemagglutinin (NTNHA) were incubated with mouse jejunum segments separately. The tissues were fixed, immunostained with a monoclonal anti-myc antibody, and visualized with a secondary antibody conjugated with Alexa Fluor 594 (red). The nuclei of the epithelial cells were labeled with DAPI (blue). The HA-wt complex showed a robust binding on luminal surface of the small intestine at both pH (A) 6.5 and (C) 7.4. In contrast, the (B) M-PTC and (D) NTNHA did not have detectable binding signal. (E) The HA-mini complex showed a similar binding pattern and intensity in comparison to the HA-wt complex. (F) The HA-DAFA mutant showed a significantly decreased binding. Images with 3× magnification were shown to the right of each panel. A minimum of three slides were prepared and analyzed for each sample, and the experiments were repeated using two mice. One slide of each sample was shown as representative. Scale bars: 0.2 mm.

Figure 3

The receptor-mimicking saccharides significantly decrease the intestinal binding of the HA complex. Mouse jejunum segments were incubated with (A) the HA-wt complex or with the same amount of complex in the presence of (B) α2,3-SiaLac, (C) α2,6-SiaLac, or (D) lactose. The HA complex and the cell nuclei were visualized as described above. All three saccharides significantly inhibited binding of the HA complex to the intestinal lumen. Scale bars: 0.2 mm.

Figure 4

Glycan array screenings with the complete HA complex and HA70. Mammalian printed array (Version 5.1) of the Consortium for Functional Glycomics (CFG) was probed with the (A) HA complex (100 μg/mL) and (B) HA70 (1 mg/mL). Ten glycans that yielded the highest binding signals are shown with their ID numbers and structures. The HA complex mostly binds to the glycans with a terminal Gal, especially the ones with a terminal Gal-GlcNAc. HA70 mostly recognizes the terminal Neu5Ac, with a preference for Neu5Ac-Gal-GlcNAc structure. Error bars: standard deviations of four measurements.