Molecular engineering of a minimal E-cadherin inhibitor protein derived from Clostridium botulinum hemagglutinin

Abstract

Hemagglutinin (HA), a nontoxic component of the botulinum neurotoxin (BoNT) complex, binds to E-cadherin and inhibits E-cadherin-mediated cell-cell adhesion. HA is a 470 kDa protein complex comprising six HA1, three HA2, and three HA3 subcomponents. Thus, to prepare recombinant full-length HA in vitro, it is necessary to reconstitute the macromolecular complex from purified HA subcomponents, which involves multiple purification steps. In this study, we developed NanoHA, a minimal E-cadherin inhibitor protein derived from Clostridium botulinum HA with a simple purification strategy needed for production. NanoHA, containing HA2 and a truncated mutant of HA3 (amino acids 380-626; termed as HA3^mini), is a 47 kDa single polypeptide (one-tenth the molecular weight of full-length HA, 470 kDa) engineered with three types of modifications: (i) a short linker sequence between the C terminus of HA2 and N terminus of HA3; (ii) a chimeric complex composed of HA2 derived from the serotype C BoNT complex and HA3^mini from the serotype B BoNT complex; and (iii) three amino acid substitutions from hydrophobic to hydrophilic residues on the protein surface. We demonstrated that NanoHA inhibits E-cadherin-mediated cell-cell adhesion of epithelial cells (e.g., Caco-2 and Madin-Darby canine kidney cells) and disrupts their epithelial barrier. Finally, unlike full-length HA, NanoHA can be transported from the basolateral side to adherens junctions via passive diffusion. Overall, these results indicate that the rational design of NanoHA provides a minimal E-cadherin inhibitor with a wide variety of applications as a lead molecule and for further molecular engineering.

Keywords: Clostridium botulinum; E-cadherin inhibitor; cell–cell adhesion; epithelial barrier disruption; hemagglutinin; iPSC culture system; molecular engineering.

Figure 1

Barrier-disrupting activities of hemagglutinin (HA)-truncated mutants.A, structure of Clostridium botulinum HA derived from the serotype B botulinum neurotoxin (BoNT) complex (Protein Data Bank [PDB] ID: 3WIN) and truncated mutants (HAΔ1; Mini-HA, construct #1; Mini-HAΔ1, construct #2). HA1, HA2, and HA3 are represented in orange, magenta, and green, respectively. B, transepithelial electrical resistance (TER) of Caco-2 cell monolayers was measured in the presence of 100 protomer nM of HA/B, HAΔ1/B, Mini-HA/B (#1), and Mini-HAΔ1/B (#2) (33.3 nM of HA/B and HAΔ1/B and 100 nM of Mini-HA/B and Mini-HAΔ1/B) at the basolateral sides. Values represent the mean ± SD of triplicate wells.

Figure 2

Molecular engineering of a single-chain CB chimeric Mini-HAΔ1 (scMini-HAΔ1/CB).A, schematic models of single-chain (sc) and chimeric Mini-HAΔ1 mutants (#2-1, #2-2, and #2-3). B, simulated model of scMini-HA containing a short linker peptide (GSGGDDPPG). C, amino acid sequence alignment of HA2/B and HA2/C. Black and green lines show the E-cadherin-binding sites and HA3-binding sites of HA2/B, respectively. The figure was generated using ClustalW (39) and the ESPript 3.0 server (40). D, SDS-PAGE of HA/B, Mini-HA/B (#1), Mini-HAΔ1/B (#2), scMini-HAΔ1/B (#2-1), Mini-HAΔ1/CB (#2-2), and scMini-HAΔ1/CB (#2-3). The gel was stained with Coomassie brilliant blue (CBB). E, transepithelial electrical resistance (TER) of Caco-2 cell monolayers was measured in the presence of 100 or 300 protomer nM of HA/B, scMini-HAΔ1/BB (#2-1), Mini-HAΔ1/CB (#2-2), and scMini-HAΔ1/CB (#2-3) at the basolateral sides. Values represent the mean ± SD of triplicate wells.

Figure 3

Molecular engineering of scMini-HAΔ1/CB-LD-YFDY (NanoHA).A, E-cadherin-binding sites are represented as gray areas on the surface model of Mini-HAΔ1/B (#2). HA2 and HA3^mini are represented in magenta and green, respectively. B, surface hydrophobicity of Mini-HAΔ1/CB. The color of the surface represents the hydrophobicity level. White, light blue, and blue represent low, medium, and high hydrophobicity, respectively. C, Leu40, Tyr73, and Phe75 of HA2 are represented as spheres on a wire model of Mini-HAΔ1/CB. D and E, TER of the Caco-2 cell monolayers was measured in the presence of 100 protomer nM of HA/B and scMini-HAΔ1/CB (WT, #2-3; YFDY, #2-3-1; LD-YFDY, #2-3-2) (D) or in the presence of HA/B (30 or 100 protomer nM; asterisks indicate protomer nM) and scMini-HAΔ1/CB-LD-YFDY (#2-3-2; NanoHA; 30, 100, 300, or 1000 nM) (E) at the basolateral side. Values represent the mean ± SD of triplicate wells. HA, hemagglutinin.

Figure 4

Binding affinity and kinetics of NanoHA for E-cadherin. Biolayer interferometry (BLI) assay was performed using a BLItz system. AMC biosensors were pre-immobilized with an anti-Strep-tag II tag antibody and then coated with Strep-tag II-tagged NanoHA (A) or Mini-HA/B (#1; B). The BLI sensorgrams were obtained using E-cadherin EC1–5 (Ecad, 30–5000 nM). Association and dissociation rate constants (K_on and K_off) and dissociation constant (K_D) were determined by global fitting to a 1:1 binding model. Red dotted lines indicate the start of the association (left) and dissociation (right) phases. HA, hemagglutinin.

Figure 5

NanoHA inhibits the cell–cell adhesion of epithelial cells. Caco-2, HT-29, CMT-93, and MDCK-I cells were cultured with vehicle (PBS), 30 nM (90 protomer nM) HA/B, or 100 nM NanoHA for 24 h and stained with Giemsa stain solution. The scale bar represents 200 μm. HA, hemagglutinin; MDCK, Madin–Darby canine kidney.

Figure 6

Binding of HAs in the lateral intercellular space at 4 °C.A and B, Caco-2 cell monolayers were incubated with His-tagged HA/B or NanoHA applied to basolateral chambers at 4 °C for 40 min. The cells were stained with anti-His-tag and anti-E-cadherin antibodies. The images show the XY planes (A) and XZ planes (B). Arrows show HAs that reside on the lateral cell surface. White dotted lines show the basal cell surface. The scale bars represent 20 μm (A) and 10 μm (B). C, proposed model of HA accessibility to the lateral intercellular space. HA/B binds to the basal surface, but not to the lateral surface, of cells at 4 °C and is transported from the basal surface to the lateral surface at 37 °C (24). Meanwhile, NanoHA binds to both surfaces at 4 °C, as shown in (A and B). HA, hemagglutinin.