In a previous study (Inui et al., 2012), we demonstrated that NTNHA and BoNT share a similar domain organization. BoNT may be divided into three domains, each of which has a distinct function, for example, the N-terminal catalytic domain (light chain; Lc), the central channel-forming domain (N-terminal half of the heavy chain; HcN), and the C-terminal binding domain (C-terminal half of the heavy chain; HcC). The C-terminal HcC binding domain of BoNT binds to nerve and intestinal epithelial cells (Maksymowych & Simpson, 2004; Stenmark et al., 2008). Based on in silico analyses (Inui et al., 2012) and crystal structures (Gu et al., 2012; Sagane et al., 2012), NTNHA and BoNT share very similar three-dimensional structures, and thus, NTNHA can also be divided into three domains: N-terminal nLc, central nHcN, and C-terminal nHcC. However, the functions of these NTNHA domains have not yet been clarified. As shown in Fig. 4(a), the nHcC domain contains both N-terminal jelly-roll and C-terminal β-trefoil domains, similar to the HcC domain of BoNT (Ginalski et al., 2000). Tetanus neurotoxin (TeNT) and serotypes A and B BoNT share the consensus sequence SXWY in the C-terminal β-trefoil domain of the HcC, which appears to be responsible for ganglioside-receptor binding on nerve cells (Rummel et al., 2004). This SXWY motif is not conserved in NTNHA, nor is it conserved in serotypes C and D BoNT (Rummel et al., 2004). These proteins share a conserved Trp residue, which is probably responsible for carbohydrate binding by the ganglioside-binding loop in the β-trefoil domain (Kroken et al., 2011). NTNHA does not possess a Trp residue in the loop region corresponding to the ganglioside-binding loop in the HcC-C domain of BoNT. On the other hand, the serotype B–F NTNHA possesses a single consensus QXW motif in its nHcC-C domain, as shown in Fig. 4(b). Additionally, serotypes A–D, and F have a conserved sequence that is a variant of the known QXW motif, QXY, 50 residues upstream from the QXW motif. On the other hand, no serotypes of BoNT possess these sequences. The QXW motif is typified by the carbohydrate-binding subunit of ricin (Hazes, 1996). Previously, Arndt et al. (2005) explained that NTNHA does not bind carbohydrates because it lacks the ganglioside-binding site that is shared by TeNT and BoNT serotypes A and B. Therefore, the function of NTNHA has been considered to be only protection of BoNT against digestive conditions. However, we found that the serotype D NTNHA molecule binds to IEC-6 cells. NTNHA also retains the β-trefoil domain and the QXW motif, which are both found in a lectin family, and the botulinum HA-33 protein also retains this motif (Hazes, 1996). Very recently, models of the three-dimensional structure of the serotypes A and B L-TC (M-TC/HAs complex) were proposed based on transmission electron microscopy images and the crystal structures of its individual components (Benefield et al., 2013). This model resembles our model for the three-dimensional structure of serotype D L-TC (Hasegawa et al., 2007), and it indicates that the C-terminal region of NTNHA, which contains candidates for the sugar-binding sites, is exposed on the surface of the molecule. Similar to the same site in NTNHA, the sugar-binding sites in HA-33, HA-70, and BoNT are fully accessible in the L-TC model (Benefield et al., 2013). The cell binding and transport through the IEC-6 cell layer of the L-TC (M-TC/HA-70/HA-17/HA-33) depend on the number of the HA-33 molecule in the TC, implying that the HA-33 enhances toxin transport through the intestinal wall (Ito et al., 2011). On the other hand, the NTNHA and HA-70 proteins do not facilitate the efficiency of the toxin transport (Ito et al., 2011). However, these multiple cell-binding sites, which have distinct binding interactions in the TC, may enhance the chances for the toxins to access different types of the intestinal epithelial cells. It is still unknown which specific residues are responsible for cell binding and transport through the intestinal epithelial cell layer. This will be addressed in future studies in which amino acid substitutions are engineered to elucidate the pathogenic mechanisms of botulinum toxin delivery in the gastrointestinal tract. Such studies should also lead to the eventual design of stable oral delivery systems for proteolytically unstable drugs.