Functional Dissection of the Clostridium botulinum Type B Hemagglutinin Complex: Identification of the Carbohydrate and E-Cadherin Binding Sites

Abstract

Botulinum neurotoxin (BoNT) inhibits neurotransmitter release in motor nerve endings, causing botulism, a condition often resulting from ingestion of the toxin or toxin-producing bacteria. BoNTs are always produced as large protein complexes by associating with a non-toxic protein, non-toxic non-hemagglutinin (NTNH), and some toxin complexes contain another non-toxic protein, hemagglutinin (HA), in addition to NTNH. These accessory proteins are known to increase the oral toxicity of the toxin dramatically. NTNH has a protective role against the harsh conditions in the digestive tract, while HA is considered to facilitate intestinal absorption of the toxin by intestinal binding and disruption of the epithelial barrier. Two specific activities of HA, carbohydrate and E-cadherin binding, appear to be involved in these processes; however, the exact roles of these activities in the pathogenesis of botulism remain unclear. The toxin is conventionally divided into seven serotypes, designated A through G. In this study, we identified the amino acid residues critical for carbohydrate and E-cadherin binding in serotype B HA. We constructed mutants defective in each of these two activities and examined the relationship of these activities using an in vitro intestinal cell culture model. Our results show that the carbohydrate and E-cadherin binding activities are functionally and structurally independent. Carbohydrate binding potentiates the epithelial barrier-disrupting activity by enhancing cell surface binding, while E-cadherin binding is essential for the barrier disruption.

Architecture of the botulinum neurotoxin complex: a molecular machine for protection and delivery

“…Consistent with this finding, HA/A binds the monomeric EC1–EC2 domains with an affinity that is much stronger than the affinity of E-cadherin homo-dimerization [40••,44,47]. The model that disruption of the adherens junctions of epithelial cells by the HA complex opens up a paracellular route to facilitate BoNT absorption has been supported by extensive in vitro and ex vivo studies [33•,40••,42] (Figure 3), and was further confirmed by an in vivo study showing that an E-cadherin binding deficient L-PTC/A has markedly decreased oral toxicity in mouse [40••]. …”

“…HA70 binds one Neu5Ac-containing carbohydrate [16••]. HA33, on the other hand, binds one galactose-containing carbohydrate through its C-terminal β-trefoil domain in serotypes A and B [16••,33•]. HA33 serotype C, however, displays a lower affinity for galactose, but carries an extra Neu5Ac-binding site near the Gal-binding pocket [30,34].…”

“…By contrast, the HAs of BoNT/C and D, which predominantly cause botulism in birds and cattle, do not bind E-cadherin [33•,40••]. Future studies should aim to understand whether or how HA/C and HA/D disrupt the intestinal epithelial barrier and the functional role of HAs in determining host tropism of various BoNTs [49,50].…”

Inhibiting oral intoxication of botulinum neurotoxin A complex by carbohydrate receptor mimics

“…The intestinal epithelial cell line Caco-2 has been widely used to characterize the transport of BoNTs in vitro (Couesnon et al, 2008; Lee et al, 2013; Matsumura et al, 2008; Sugawara et al, 2014). In a trans-well assay, Caco-2 cells are differentiated on a permeable poly-carbonate support to form a polarized columnar cell monolayer resembling the small intestinal epithelial layer with high transepithelial resistance against passive diffusion of ion and solutes.…”

“…HA70 and HA33 carry an N-acetylneuraminic acid (Neu5Ac) and a galactose (Gal) binding site, respectively. Therefore, each HA complex comprises a total of nine glycan-binding sites, which allow multivalent interactions with host carbohydrates to enrich the toxin complex on the intestinal surface (Lee et al, 2013; Matsumura et al, 2015; Sugawara et al, 2014; Yao et al, 2014). The HA complex then interacts with E-cadherin, a major host adhesion protein, to disrupt the E-cadherin mediated cell-cell adhesion and open the paracellular route facilitating the transepithelial delivery of BoNT (Lee et al, 2014b; Matsumura et al, 2008; Sugawara et al, 2010).…”