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

Open AccessPublished:January 24, 2023DOI:https://doi.org/10.1016/j.jbc.2023.102944

      Abstract

      Hemagglutinin (HA), a non-toxic 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 (aa 380-626; termed as HA3mini), 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 HA3mini 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 MDCK 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

      Abbreviations:

      BoNT (botulinum neurotoxin), HA (hemagglutinin), AJs (adherens junctions), aa (amino acid), KD (dissociation constant), Kon (association rate constant), Koff (dissociation rate constant), TER (transepithelial electrical resistance), sc (single-chain)

      Introduction

      Clostridium botulinum produces the botulinum neurotoxin (BoNT) complex, one of the most potent natural toxins causing botulism (
      • Schiavo G.
      • Matteoli M.
      • Montecucco C.
      Neurotoxins affecting neuroexocytosis.
      ). The BoNT complex comprises neurotoxin (BoNT), non-toxic non-hemagglutinin (NTNHA), and hemagglutinin (HA) (
      • Sakaguchi G.
      Clostridium botulinum toxins.
      ). BoNTs are traditionally classified into seven serotypes (A–G) (
      • Schiavo G.
      • Matteoli M.
      • Montecucco C.
      Neurotoxins affecting neuroexocytosis.
      ). HA is a non-toxic 470 kDa protein complex comprising six HA1, three HA2, and three HA3 subcomponents (
      • Amatsu S.
      • Sugawara Y.
      • Matsumura T.
      • Kitadokoro K.
      • Fujinaga Y.
      Crystal structure of Clostridium botulinum whole hemagglutinin reveals a huge triskelion-shaped molecular complex.
      ). HA has at least two biological activities that promote intestinal absorption of BoNTs: carbohydrate-binding activity (
      • Fujinaga Y.
      • Inoue K.
      • Watanabe S.
      • Yokota K.
      • Hirai Y.
      • Nagamachi E.
      • Oguma K.
      The haemagglutinin of Clostridium botulinum type C progenitor toxin plays an essential role in binding of toxin to the epithelial cells of guinea pig small intestine, leading to the efficient absorption of the toxin.
      ,
      • Fujinaga Y.
      • Inoue K.
      • Nomura T.
      • Sasaki J.
      • Marvaud J.C.
      • Popoff M.R.
      • Kozaki S.
      • Oguma K.
      Identification and characterization of functional subunits of Clostridium botulinum type A progenitor toxin involved in binding to intestinal microvilli and erythrocytes.
      ,
      • Fujinaga Y.
      • Inoue K.
      • Watarai S.
      • Sakaguchi Y.
      • Arimitsu H.
      • Lee J.-C.
      • Jin Y.
      • Matsumura T.
      • Kabumoto Y.
      • Watanabe T.
      • Ohyama T.
      • Nishikawa A.
      • Oguma K.
      Molecular characterization of binding subcomponents of Clostridium botulinum type C progenitor toxin for intestinal epithelial cells and erythrocytes.
      ,
      • Arimitsu H.
      • Sakaguchi Y.
      • Lee J.-C.
      • Ochi S.
      • Tsukamoto K.
      • Yamamoto Y.
      • Ma S.
      • Tsuji T.
      • Oguma K.
      Molecular properties of each subcomponent in Clostridium botulinum type B haemagglutinin complex.
      ,
      • Yao G.
      • Lee K.
      • Gu S.
      • Lam K.-H.
      • Jin R.
      Botulinum neurotoxin A complex recognizes host carbohydrates through its hemagglutinin component.
      ,
      • Matsumura T.
      • Sugawara Y.
      • Yutani M.
      • Amatsu S.
      • Yagita H.
      • Kohda T.
      • Fukuoka S.-I.
      • Nakamura Y.
      • Fukuda S.
      • Hase K.
      • Ohno H.
      • Fujinaga Y.
      Botulinum toxin A complex exploits intestinal M cells to enter the host and exert neurotoxicity.
      ,
      • Lam T.I.
      • Stanker L.H.
      • Lee K.
      • Jin R.
      • Cheng L.W.
      Translocation of botulinum neurotoxin serotype A and associated proteins across the intestinal epithelia.
      ,
      • Amatsu S.
      • Fujinaga Y.
      Botulinum hemagglutinin: critical protein for adhesion and absorption of neurotoxin complex in host intestine.
      ) and E-cadherin-binding activity (
      • Matsumura T.
      • Jin Y.
      • Kabumoto Y.
      • Takegahara Y.
      • Oguma K.
      • Lencer W.I.
      • Fujinaga Y.
      The HA proteins of botulinum toxin disrupt intestinal epithelial intercellular junctions to increase toxin absorption.
      ,
      • Sugawara Y.
      • Matsumura T.
      • Takegahara Y.
      • Jin Y.
      • Tsukasaki Y.
      • Takeichi M.
      • Fujinaga Y.
      Botulinum hemagglutinin disrupts the intercellular epithelial barrier by directly binding E-cadherin.
      ,
      • Lee K.
      • Zhong X.
      • Gu S.
      • Kruel A.M.
      • Dorner M.B.
      • Perry K.
      • Rummel A.
      • Dong M.
      • Jin R.
      Molecular basis for disruption of E-cadherin adhesion by botulinum neurotoxin A complex.
      ). HAs derived from the serotype A BoNT complex (HA/A) and serotype B BoNT complex (HA/B) bind to the EC1-2 domain of E-cadherin, inhibiting its dimerization and causing epithelial barrier disruption (
      • Matsumura T.
      • Jin Y.
      • Kabumoto Y.
      • Takegahara Y.
      • Oguma K.
      • Lencer W.I.
      • Fujinaga Y.
      The HA proteins of botulinum toxin disrupt intestinal epithelial intercellular junctions to increase toxin absorption.
      ,
      • Sugawara Y.
      • Matsumura T.
      • Takegahara Y.
      • Jin Y.
      • Tsukasaki Y.
      • Takeichi M.
      • Fujinaga Y.
      Botulinum hemagglutinin disrupts the intercellular epithelial barrier by directly binding E-cadherin.
      ).
      E-cadherin is an important cell-adhesion molecule that mediates cell-cell adhesion at adherens junctions (AJs) in epithelial cells (
      • Takeichi M.
      Cadherin cell adhesion receptors as a morphogenetic regulator.
      ,
      • van Roy F.
      • Berx G.
      The cell-cell adhesion molecule E-cadherin.
      ). AJs are located beneath tight junctions (TJs), which prevent diffusion of molecules between apical and basolateral membranes (
      • Zihni C.
      • Mills C.
      • Matter K.
      • Balda M.S.
      Tight junctions: from simple barriers to multifunctional molecular gates.
      ). Type I classic cadherins, including E-cadherin, contain five extracellular domains (EC1–5), a single-transmembrane domain, and an intracellular domain. The EC1-2 domains of type I classic cadherins bind another cadherin to form trans-dimer and cis-dimer in a calcium ion-dependent manner. E-cadherin-mediated cell-cell adhesion plays a pivotal role in the development and maintenance of tissue organization. E-cadherin-mediated cell contacts are known to inhibit cell proliferation through the Hippo signaling pathway (
      • Kim N.-G.
      • Koh E.
      • Chen X.
      • Gumbiner B.M.
      E-cadherin mediates contact inhibition of proliferation through Hippo signaling-pathway components.
      ). HA/A and HA/B inhibit E-cadherin-mediated cell contacts (
      • Matsumura T.
      • Jin Y.
      • Kabumoto Y.
      • Takegahara Y.
      • Oguma K.
      • Lencer W.I.
      • Fujinaga Y.
      The HA proteins of botulinum toxin disrupt intestinal epithelial intercellular junctions to increase toxin absorption.
      ,
      • Sugawara Y.
      • Matsumura T.
      • Takegahara Y.
      • Jin Y.
      • Tsukasaki Y.
      • Takeichi M.
      • Fujinaga Y.
      Botulinum hemagglutinin disrupts the intercellular epithelial barrier by directly binding E-cadherin.
      ,
      • Jin Y.
      • Takegahara Y.
      • Sugawara Y.
      • Matsumura T.
      • Fujinaga Y.
      Disruption of the epithelial barrier by botulinum haemagglutinin (HA) proteins - differences in cell tropism and the mechanism of action between HA proteins of types A or B, and HA proteins of type C.
      ) and promote the proliferation of Caco-2 cells (human colon carcinoma-derived epithelial cell line), T84 cells (human colon carcinoma-derived epithelial cell line), and MDCK-I cells (canine kidney epithelial cell line) (
      • Jin Y.
      • Takegahara Y.
      • Sugawara Y.
      • Matsumura T.
      • Fujinaga Y.
      Disruption of the epithelial barrier by botulinum haemagglutinin (HA) proteins - differences in cell tropism and the mechanism of action between HA proteins of types A or B, and HA proteins of type C.
      ). Further, HA disperses human induced pluripotent stem (iPS) cell (hiPSC) aggregates in 3D suspension culture, resulting in a larger number of live cells, higher cell density, and higher-fold expansion than those of cells treated with conventional digestive enzymes (
      • Nath S.C.
      • Tokura T.
      • Kim M.-H.
      • Kino-oka M.
      Botulinum hemagglutinin-mediated in situ break-up of human induced pluripotent stem cell aggregates for high-density suspension culture.
      ). Therefore, hiPSC culture systems with HA facilitate simple and robust maintenance of undifferentiated cells (
      • Nath S.C.
      • Tokura T.
      • Kim M.-H.
      • Kino-oka M.
      Botulinum hemagglutinin-mediated in situ break-up of human induced pluripotent stem cell aggregates for high-density suspension culture.
      ,
      • Kim M.-H.
      • Matsubara Y.
      • Fujinaga Y.
      • Kino-oka M.
      A simple and robust method for culturing human-induced pluripotent stem cells in an undifferentiated state using botulinum hemagglutinin.
      ,
      • Kim M.-H.
      • Sugawara Y.
      • Fujinaga Y.
      • Kino-oka M.
      Botulinum hemagglutinin-mediated selective removal of cells deviating from the undifferentiated state in hiPSC colonies.
      ).
      HA forms a heterododecameric complex that adopts a large triskelion-shaped structure (Fig. 1A) (
      • Amatsu S.
      • Sugawara Y.
      • Matsumura T.
      • Kitadokoro K.
      • Fujinaga Y.
      Crystal structure of Clostridium botulinum whole hemagglutinin reveals a huge triskelion-shaped molecular complex.
      ). In a previous study, the recombinant HA/B complex was reconstituted in vitro, by mixing the recombinant proteins of HA1, HA2, and HA3 and incubating at 37°C for 3 hr (
      • Amatsu S.
      • Sugawara Y.
      • Matsumura T.
      • Kitadokoro K.
      • Fujinaga Y.
      Crystal structure of Clostridium botulinum whole hemagglutinin reveals a huge triskelion-shaped molecular complex.
      ). Thus, three protein expression systems (HA1, HA2, and HA3) and four purification steps (three HA subcomponents and in vitro reconstitution) are required to obtain the HA/B complex.
      Figure thumbnail gr1
      Figure1Barrier-disrupting activities of hemagglutinin (HA)-truncated mutants. A. Structure of C. botulinum HA derived from the serotype B BoNT complex (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.
      In the present study, we developed NanoHA, a minimal E-cadherin inhibitor protein derived from HA (termed NanoHA). NanoHA inhibits cell-cell adhesion and disrupts the epithelial barrier, similar to full-length wild-type HA. Further, NanoHA has one-tenth the molecular weight of full-length HA and can be purified in sufficient quantities using a simple purification strategy.

      Results

      Minimization of HA

      To identify the essential HA fragments that disrupt the epithelial barrier, we tested the barrier-disrupting activity of full-length HA/B (HA/B: HA1/B+HA2/B+HA3/B), HA/B lacking HA1/B (HAΔ1/B: HA2/B+HA3/B), HA/B lacking the trimerization domain of HA3/B (Mini-HA/B: HA1/B+HA2/B+HA3mini/B, construct #1) (
      • Lee K.
      • Gu S.
      • Jin L.
      • Le T.T.N.
      • Cheng L.W.
      • Strotmeier J.
      • Kruel A.M.
      • Yao G.
      • Perry K.
      • Rummel A.
      • Jin R.
      Structure of a bimodular botulinum neurotoxin complex provides insights into its oral toxicity.
      ), and Mini-HA/B lacking HA1/B (Mini-HAΔ1/B: HA2/B+HA3mini/B, construct #2) (Fig. 1A and 2D and Fig. S1). Within 4 hr post-addition, 100 (protomer) nM HA/B, HAΔ1/B, and Mini-HA/B (#1) reduced the transepithelial electrical resistance (TER) of the Caco-2 cell monolayer to 25% of that at 0 hr post-addition, whereas 100 nM of Mini-HAΔ1/B (#2) did not affect the TER (Fig. 1B). In this study, we calculated protein concentration in protomer units for HA/B and HAΔ1/B, because HA comprises three protomers (
      • Amatsu S.
      • Sugawara Y.
      • Matsumura T.
      • Kitadokoro K.
      • Fujinaga Y.
      Crystal structure of Clostridium botulinum whole hemagglutinin reveals a huge triskelion-shaped molecular complex.
      ,
      • Lee K.
      • Gu S.
      • Jin L.
      • Le T.T.N.
      • Cheng L.W.
      • Strotmeier J.
      • Kruel A.M.
      • Yao G.
      • Perry K.
      • Rummel A.
      • Jin R.
      Structure of a bimodular botulinum neurotoxin complex provides insights into its oral toxicity.
      ) and each protomer has one E-cadherin-binding site (
      • Lee K.
      • Zhong X.
      • Gu S.
      • Kruel A.M.
      • Dorner M.B.
      • Perry K.
      • Rummel A.
      • Dong M.
      • Jin R.
      Molecular basis for disruption of E-cadherin adhesion by botulinum neurotoxin A complex.
      ): i.e., 1 nM HA/B is equal to 3 protomer nM HA/B.
      Figure thumbnail gr2
      Figure 2Molecular 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, #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 (
      • Thompson J.D.
      • Higgins D.G.
      • Gibson T.J.
      CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.
      ) and the ESPript 3.0 server (
      • Robert X.
      • Gouet P.
      Deciphering key features in protein structures with the new ENDscript server.
      ). 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.

      Engineering of Mini-HAΔ1

      Mini-HA (#1) is observed to binds to E-cadherin with a dissociation constant (KD) similar to that of HA (∼2.7 μM vs. ∼2.3 μM) (
      • Lee K.
      • Zhong X.
      • Gu S.
      • Kruel A.M.
      • Dorner M.B.
      • Perry K.
      • Rummel A.
      • Dong M.
      • Jin R.
      Molecular basis for disruption of E-cadherin adhesion by botulinum neurotoxin A complex.
      ), and disrupts the epithelial barrier (Fig. 1B) (
      • Lee K.
      • Zhong X.
      • Gu S.
      • Kruel A.M.
      • Dorner M.B.
      • Perry K.
      • Rummel A.
      • Dong M.
      • Jin R.
      Molecular basis for disruption of E-cadherin adhesion by botulinum neurotoxin A complex.
      ,
      • Amatsu S.
      • Matsumura T.
      • Yutani M.
      • Fujinaga Y.
      Multivalency effects of hemagglutinin component of type B botulinum neurotoxin complex on epithelial barrier disruption.
      ). HAΔ1 also binds to E-cadherin and disrupts the epithelial barrier (Fig. 1B) (
      • Amatsu S.
      • Sugawara Y.
      • Matsumura T.
      • Kitadokoro K.
      • Fujinaga Y.
      Crystal structure of Clostridium botulinum whole hemagglutinin reveals a huge triskelion-shaped molecular complex.
      ,
      • Sugawara Y.
      • Matsumura T.
      • Takegahara Y.
      • Jin Y.
      • Tsukasaki Y.
      • Takeichi M.
      • Fujinaga Y.
      Botulinum hemagglutinin disrupts the intercellular epithelial barrier by directly binding E-cadherin.
      ). These data led us to hypothesize that Mini-HAΔ1 (#2) has potential to disrupt the epithelial barrier, even though Mini-HAΔ1/B failed to do so (Fig. 1B). First, we attempted to improve the barrier-disrupting activity of the HA2/B+HA3mini/B complex (Mini-HAΔ1, #2). As single-chain fragment variable (scFv) fragments are known to be more stable than their Fv fragments (
      • Wörn A.
      • Plückthun A.
      Stability engineering of antibody single-chain Fv fragments.
      ), we designed and tested single-chain Mini-HAΔ1/B (scMini-HAΔ1/B, construct #2-1). Previously, we determined the crystal structure of full-length HA/B (
      • Amatsu S.
      • Sugawara Y.
      • Matsumura T.
      • Kitadokoro K.
      • Fujinaga Y.
      Crystal structure of Clostridium botulinum whole hemagglutinin reveals a huge triskelion-shaped molecular complex.
      ). To preserve the spatial arrangement between HA2/B and HA3/B, we fused the N-terminus of HA2/B and the C-terminus of HA3mini/B with a short linker sequence (scMini-HAΔ1/B, #2-1). (Fig. 2A, B and D and Fig. S1). We found that 100 nM and 300 nM of scMini-HAΔ1/B (#2-1) reduced the TER of Caco-2 monolayers to 76% and 49% compared with that at 0 hr post-addition, respectively (Fig. 2E). However, they did not reduce the TER completely, whereas 100 protomer nM of HA/B did (Fig. 2E). These data indicate that fusing HA2 and HA3 via a short linker maintains the structural integrity of the natural noncovalent interaction between HA2 and HA3. Next, we replaced HA2/B in Mini-HAΔ1/B with HA2/C (Mini-HAΔ1/CB, construct #2-2) (Fig. 2A, C, and D and Fig. S1). HA2/B shows 98% and 64% amino acid sequence homology with HA2/A and HA2/C, respectively. We previously demonstrated that a BCB chimeric full-length HA complex (HA1/B+HA2/C+HA3/B) binds to E-cadherin in a manner similar to HA/B, whereas a BBC chimeric complex (HA1/B+HA2/B+HA3/C) does not (
      • Sugawara Y.
      • Yutani M.
      • Amatsu S.
      • Matsumura T.
      • Fujinaga Y.
      Functional dissection of the Clostridium botulinum Type B hemagglutinin complex: identification of the carbohydrate and E-cadherin binding sites.
      ). Treatment with 100 nM and 300 nM Mini-HAΔ1/CB (#2-2) reduced TER to 81% and 46% of that at 0 hr post-addition, respectively (Fig. 2E). These TER values were recovered at 4 hr post-addition (Fig. 2E). Although chimerization of HA2 does not largely affect the E-cadherin-binding activity itself (
      • Sugawara Y.
      • Yutani M.
      • Amatsu S.
      • Matsumura T.
      • Fujinaga Y.
      Functional dissection of the Clostridium botulinum Type B hemagglutinin complex: identification of the carbohydrate and E-cadherin binding sites.
      ), it may increase barrier-disrupting activity via indirect effects such as multimerization (
      • Amatsu S.
      • Matsumura T.
      • Yutani M.
      • Fujinaga Y.
      Multivalency effects of hemagglutinin component of type B botulinum neurotoxin complex on epithelial barrier disruption.
      ) or stabilization of the truncated mutant. A single-chain CB chimeric Mini-HAΔ1 (scMini-HAΔ1/CB, construct #2-3) further reduced the TER to 38% at 100 nM and 29% at 300 nM of that at 0 hr post-addition (Fig. 2E).

      Engineering of NanoHA

      Hydrophobic to hydrophilic amino acid mutations on the protein surface improve protein solubility (
      • Trevino S.R.
      • Scholtz J.M.
      • Pace C.N.
      Amino acid contribution to protein solubility: Asp, Glu, and Ser contribute more favorably than the other hydrophilic amino acids in RNase Sa.
      ). To further improve the barrier-disrupting activity, we introduced amino acid substitutions in the surface region of scMini-HAΔ1/CB, except for the E-cadherin-binding sites, to decrease hydrophobicity (Fig. 3A−C and Fig. S1). We found that scMini-HAΔ1/CB with Tyr73Asp and Phe75Tyr mutations in HA2/C (YFDY, construct #2-3-1) decreased the TER of the Caco-2 cell monolayer more effectively than wild-type scMini-HAΔ1/CB (WT) (27% vs. 39%) (Fig. 3D). Furthermore, scMini-HAΔ1/CB-YFDY with a Leu40Asp mutation in HA2/C (LD-YFDY, construct #2-3-2) decreased TER more effectively than did the YFDY mutant (18% vs. 27%) (Fig. 3D). We found that 100 nM and 300 nM of scMini-HAΔ1/CB-LD-YFDY (#2-3-2, termed NanoHA) exhibited barrier-disrupting activity similar to that of 30 protomer nM and 100 protomer nM of HA/B, respectively (Fig. 3E). These results indicate that the HA2+HA3mini complex is the intrinsic core component essential for HA to inhibit E-cadherin and disrupt the epithelial barrier.
      Figure thumbnail gr3
      Figure 3Molecular 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 HA3mini 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.

      Binding affinity and kinetics for E-cadherin

      NanoHA binds to E-cadherin with lower affinity (KD ∼ 2.6 μM) than Mini-HA/B (#1; KD ∼ 0.12 μM) (Fig. 4). The association rate constant (Kon) is similar between NanoHA (1.8 × 10-4 Ms-1) and Mini-HA/B (1.6 × 10-4 Ms-1), whereas NanoHA has a faster dissociation rate constant (Koff) than Mini-HA/B (4.8 × 10-2 s-1 vs. 0.19 × 10-2 s-1). Thus, the difference in binding affinity (KD) is attributed to a faster Koff of NanoHA. These results suggest that the fast Koff affects the barrier-disrupting activity of NanoHA.
      Figure thumbnail gr4
      Figure 4Binding affinity and kinetics of NanoHA for E-cadherin. Bio-layer 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, 30–5,000 nM). Association and dissociation rate constants (Kon and Koff) and dissociation constant (KD) 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.

      Inhibition of cell-cell adhesion

      To test the effect of NanoHA on cell-cell adhesion in different cell lines, we added NanoHA to Caco-2, HT-29 (human colon carcinoma-derived epithelial cell line), CMT-93 (mouse rectum carcinoma-derived epithelial cell line), and MDCK-I cells. We found that NanoHA inhibited cell-cell adhesion similarly to HA/B (Fig. 5 and Fig. S2). In particular, the colonies of HT-29, CMT-93, and MDCK-I cells were dispersed by these treatments (Fig. 5 and Fig. S2). The treated cells largely remained attached to the culture plates (Fig. 5 and Fig. S2), suggesting that HA and NanoHA did not inhibit cell-matrix adhesion.
      Figure thumbnail gr5
      Figure 5NanoHA 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 hr, and stained with Giemsa Stain Solution. Scale bar: 200 μm.

      Binding to the basolateral cell surface at 4°C

      To approach AJs from the basolateral side, basolaterally applied HA/B initially binds to the basal surface of Caco-2 cells. The cell-bound HA/B is then transported to the lateral surface of the cell in an E-cadherin-binding ability-dependent manner (
      • Amatsu S.
      • Matsumura T.
      • Yutani M.
      • Fujinaga Y.
      Multivalency effects of hemagglutinin component of type B botulinum neurotoxin complex on epithelial barrier disruption.
      ). We propose that cadherin-flow (
      • Kametani Y.
      • Takeichi M.
      Basal-to-apical cadherin flow at cell junctions.
      ) transports HA from the basal to lateral surface. NanoHA comprises the core components of HA, which are essential to inhibit E-cadherin and has one-tenth molecular weight (47 kDa vs. 488 kDa) and one-fourth molecular size (65 Å vs. 280 Å diameter) compared to that of full-length HA (Fig. 1A and 6C ). Therefore, we hypothesized that NanoHA is advantageous in terms of accessibility to intercellular spaces. To test this hypothesis, we added HAs to the basolateral sides of Caco-2 cell monolayers and incubated them at 4°C. Consequently, NanoHA was localized to both the basal and lateral cell surfaces (Fig. 6A and B), whereas HA/B bound only to the basal surface (Fig. 6A and B) (
      • Amatsu S.
      • Matsumura T.
      • Yutani M.
      • Fujinaga Y.
      Multivalency effects of hemagglutinin component of type B botulinum neurotoxin complex on epithelial barrier disruption.
      ). This indicates that NanoHA can access AJs by passive diffusion, and that HA/B is too large to penetrate lateral intercellular spaces (Fig. 6C).
      Figure thumbnail gr6
      Figure 6Binding 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. Scale bars: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 (
      • Amatsu S.
      • Matsumura T.
      • Yutani M.
      • Fujinaga Y.
      Multivalency effects of hemagglutinin component of type B botulinum neurotoxin complex on epithelial barrier disruption.
      ). Meanwhile, NanoHA binds to both surfaces at 4°C, as shown in (A and B).

      Discussion

      In this study, we developed a minimal E-cadherin inhibitor protein, NanoHA, derived from C. botulinum HA through rational design. scMini-HAΔ1/CB-LD-YFDY (construct #2-3-2, termed NanoHA) has three types of modifications: a short peptide linker to preserve structural integrity, chimerization, and three point mutations (Figure 2, Figure 3B, C and Fig. S1). NanoHA inhibited cell-cell contacts and disrupted the epithelial barrier (Fig. 3E and 5 and Fig. S2). To obtain high-quality purified recombinant protein, full-length HA needs four purification steps: purification of HA1, HA2, and HA3 and full-length HA after in vitro reconstitution. In contrast, NanoHA can be purified in a single step and shows a 10-fold higher protein yield than that of full-length HA (data not shown).
      Cadherin-mediated cell-cell adhesions are abrogated by other cadherin inhibitors, such as ADH-1 (also known as ExherinTM), a cyclic peptide composed of the His-Ala-Val (HAV) sequence of N-cadherin (
      • Williams E.
      • Williams G.
      • Gour B.J.
      • Blaschuk O.W.
      • Doherty P.
      A novel family of cyclic peptide antagonists suggests that N-cadherin specificity is determined by amino acids that flank the HAV motif.
      ); Epep, an E-cadherin mimic peptide H-SWELYYPLRANL-NH2 (
      • Devemy E.
      • Blaschuk O.W.
      Identification of a novel dual E- and N-cadherin antagonist.
      ); and E-cadherin-neutralizing antibodies (
      • Qian X.
      • Karpova T.
      • Sheppard A.M.
      • McNally J.
      • Lowy D.R.
      E-cadherin-mediated adhesion inhibits ligand-dependent activation of diverse receptor tyrosine kinases.
      ,
      • Ohgushi M.
      • Matsumura M.
      • Eiraku M.
      • Murakami K.
      • Aramaki T.
      • Nishiyama A.
      • Muguruma K.
      • Nakano T.
      • Suga H.
      • Ueno M.
      • Ishizaki T.
      • Suemori H.
      • Narumiya S.
      • Niwa H.
      • Sasai Y.
      Molecular pathway and cell state responsible for dissociation-induced apoptosis in human pluripotent stem cells.
      ). These E-cadherin inhibitors induce apoptosis in some cell lines by abrogating cell-cell contact (
      • Shintani Y.
      • Fukumoto Y.
      • Chaika N.
      • Grandgenett P.M.
      • Hollingsworth M.A.
      • Wheelock M.J.
      • Johnson K.R.
      ADH-1 suppresses N-cadherin-dependent pancreatic cancer progression.
      ,
      • Hawkins K.
      • Mohamet L.
      • Ritson S.
      • Merry C.L.R.
      • Ward C.M.
      E-cadherin and, in its absence, N-cadherin promotes Nanog expression in mouse embryonic stem cells via STAT3 phosphorylation.
      ,
      • Ohgushi M.
      • Matsumura M.
      • Eiraku M.
      • Murakami K.
      • Aramaki T.
      • Nishiyama A.
      • Muguruma K.
      • Nakano T.
      • Suga H.
      • Ueno M.
      • Ishizaki T.
      • Suemori H.
      • Narumiya S.
      • Niwa H.
      • Sasai Y.
      Molecular pathway and cell state responsible for dissociation-induced apoptosis in human pluripotent stem cells.
      ). In contrast, HA/A and HA/B are not toxic to epithelial cells, such as Caco-2 cells and MDCK-I cells, but rather promote cell proliferation (
      • Jin Y.
      • Takegahara Y.
      • Sugawara Y.
      • Matsumura T.
      • Fujinaga Y.
      Disruption of the epithelial barrier by botulinum haemagglutinin (HA) proteins - differences in cell tropism and the mechanism of action between HA proteins of types A or B, and HA proteins of type C.
      ). NanoHA also does not affect cell viability of Caco-2, HT-29, CMT-93, MDCK, and HeLa cells (Fig. S3). Recently, we reported a simple and robust method to maintain iPS cells in an undifferentiated state using full-length HA (
      • Nath S.C.
      • Tokura T.
      • Kim M.-H.
      • Kino-oka M.
      Botulinum hemagglutinin-mediated in situ break-up of human induced pluripotent stem cell aggregates for high-density suspension culture.
      ,
      • Kim M.-H.
      • Matsubara Y.
      • Fujinaga Y.
      • Kino-oka M.
      A simple and robust method for culturing human-induced pluripotent stem cells in an undifferentiated state using botulinum hemagglutinin.
      ,
      • Kim M.-H.
      • Sugawara Y.
      • Fujinaga Y.
      • Kino-oka M.
      Botulinum hemagglutinin-mediated selective removal of cells deviating from the undifferentiated state in hiPSC colonies.
      ). We demonstrated that NanoHA, as well as full-length HA, disrupts the epithelial barrier (Fig. 3E) and inhibits E-cadherin-mediated cell-cell contacts in epithelial cells (Fig. 5 and Fig. S2). Thus, NanoHA can be used in novel iPSC culture systems. NanoHA (47 kDa) has one-tenth the molecular weight of full-length HA (native HA, 470 kDa; recombinant HA including affinity tags, 488 kDa) (Fig. 1A) and is favorable for penetrating intercellular spaces (Fig. 6). Therefore, NanoHA has the advantage of being an E-cadherin inhibitor for tightly packed 3D-cultured iPSC aggregates compared to full-length HA. Further, NanoHA provides a wide variety of applications as a useful basal protein tool for further molecular engineering, such as directed evolution using phage display (
      • Brödel A.K.
      • Isalan M.
      • Jaramillo A.
      Engineering of biomolecules by bacteriophage directed evolution.
      ), and addition of other protein components (
      • Tateno H.
      • Onuma Y.
      • Ito Y.
      • Minoshima F.
      • Saito S.
      • Shimizu M.
      • Aiki Y.
      • Asashima M.
      • Hirabayashi J.
      Elimination of tumorigenic human pluripotent stem cells by a recombinant lectin-toxin fusion protein.
      ) or functional signal peptides such as cell-targeting peptides (
      • Liu M.
      • Fang X.
      • Yang Y.
      • Wang C.
      Peptide-enabled targeted delivery systems for therapeutic applications.
      ).

      Experimental procedures

      Plasmid construction

      Genomic DNA was extracted and purified from C. botulinum serotype B strain Okra and serotype C strain Stockholm. HA1 (aa 7-294) derived from the serotype B BoNT complex (HA1/B)-encoding gene, excluding the stop codon, was cloned into the NheI-SalI site of the pET28b(+) vector (Merck), and an oligonucleotide encoding a FLAG-tag was inserted at the C-terminus of HA1/B. HA2 (aa 2-146) derived from the serotype B BoNT complex (HA2/B) or serotype C BoNT complex (HA2/C) encoding gene was cloned into the NheI-SalI site of the pET28b(+) vector. Full-length HA3 (aa 19-626) derived from the serotype B BoNT complex (HA3/B)-encoding gene, excluding the stop codon, was cloned into the NcoI-SalI site of the pET52b(+) vector (Merck). The truncated mutant of HA3 (termed HA3mini, aa 380-626) derived from the serotype B BoNT complex (HA3mini/B)-encoding gene, excluding the stop codon, was cloned into the NcoI-SalI site of the pET52b(+) vector. An oligonucleotide encoding a Strep-tag II tag was inserted at the C-termini of HA3 and HA3mini. For single-chain Mini-HAΔ1 (scMini-HAΔ1) proteins, HA3mini, an oligonucleotide encoding a short linker sequence (GSGGDDPPG), HA2, and an oligonucleotide encoding a Strep-tag II tag were cloned into the NcoI-SalI site of the pET52b(+) vector. Site-directed mutagenesis was performed using PrimeSTAR Max Polymerase (TaKaRa Bio). The inserted regions of these plasmids and the presence of mutations were confirmed by DNA sequencing.

      Protein expression and purification

      Rosetta2 (DE3) E. coli cells (Merck) were grown in Terrific Broth (TB) medium. The expression of HA proteins was induced using Overnight Express Autoinduction System 1 (Merck) at 18°C for 48 hr. Cells were harvested and lysed in PBS (pH 7.4) by sonication. His-HA1-FLAG, His-HA2, and His-NanoHA were purified using HisTrap HP (Cytiva). Strep-tag II-tagged proteins were purified using StrepTrap HP (Cytiva). These column purification steps were performed following manufacturer’s protocols. All proteins were dialyzed against PBS (pH 7.4) and stored at -80°C until further analysis. The protein concentration of the samples was determined using the Pierce BCA assay (Thermo Fisher Scientific).

      In vitro reconstitution and purification

      The HA protein complexes were reconstituted and purified as previously described (
      • Amatsu S.
      • Sugawara Y.
      • Matsumura T.
      • Kitadokoro K.
      • Fujinaga Y.
      Crystal structure of Clostridium botulinum whole hemagglutinin reveals a huge triskelion-shaped molecular complex.
      ). For HA and Mini-HA, purified HA1, HA2, and HA3 (or HA3mini) were mixed at a molar ratio of 4:4:1. For HAΔ1 and Mini-HAΔ1, purified HA2 and HA3 (or HA3mini) were mixed at a molar ratio of 4:1.

      Transepithelial electrical resistance assay

      TER was measured using Millicell-ERS (Merck) as previously described (
      • Matsumura T.
      • Jin Y.
      • Kabumoto Y.
      • Takegahara Y.
      • Oguma K.
      • Lencer W.I.
      • Fujinaga Y.
      The HA proteins of botulinum toxin disrupt intestinal epithelial intercellular junctions to increase toxin absorption.
      ). Briefly, HAs were added to the basolateral chambers of a Transwell (Corning) with the Caco-2 cell monolayer, and the plates were incubated at 37°C with 5% CO2. TER was measured at time points up to 24 or 72 hr post-addition.

      Optical microscopy

      Caco-2, HT-29, CMT-93, and MDCK cells were incubated with 0–100 (protomer) nM of HA/B or NanoHA at 37°C with 5% CO2 for 24 hr in 24-well plates (Iwaki). The plates were stained with Giemsa Stain Solution (FUJIFILM Wako Chemicals) according to the manufacturer’s protocol and observed using a BZ-X700 all-in-one fluorescence microscope (KEYENCE).

      Immunofluorescence

      The basolateral side of Caco-2 cells grown on a Transwell was treated with 17 nM (51 protomer nM) of HA/B (His-HA1/B-FLAG+His-HA2/B+HA3/B-strep) or 51 nM of His-NanoHA were added to the basolateral chamber of Transwell with the Caco-2 cell monolayer. The plates were then incubated at 4°C for 40 min. After washing, the cells on Transwell filter membranes were fixed with 4% PFA at room temperature for 15 min, permeabilized with 0.5% Triton X-100/PBS at room temperature for 5 min, and blocked with 5% BSA/PBS. The cells were then incubated with mouse anti-His-tag monoclonal antibody (OGHis, MBL; 1:1000) and rat anti-E-cadherin monoclonal antibody (DECMA-1, Merck; 1:1000), followed by Alexa Fluor 488-conjugated anti-mouse IgG antibody (Thermo Fisher Scientific; 1:400) and Cy3-conjugated anti-rat IgG antibody (Jackson ImmunoResearch; 1:400). The slides were mounted with ProLong Diamond Antifade mountant. Images were acquired by confocal microscopy using an IX71 microscope (Olympus) and a CSUX1 confocal scanner unit (Yokogawa Electric), and processed using Metamorph software (Molecular Devices).

      Structure modeling of NanoHA

      The molecular model of MiniHAΔ1/CB was built by manually docking the crystal structures of HA2 from the HA1/D-HA2/D complex (PDB ID: 2E4M) onto that of HA/B (PDB ID: 3WIN), as HA2/D shows 99% amino acid sequence homology with HA2/C. A short peptide linker sequence, Gly-Ser-Gly-Gly-Asp-Asp-Pro-Pro-Gly, was inserted between the C-terminus of HA3mini/B and the N-terminus of HA2/D, and the model was refined by simulated annealing using GROMACS (
      • Abraham M.J.
      • Murtola T.
      • Schulz R.
      • Páll S.
      • Smith J.C.
      • Hess B.
      • Lindahl E.
      GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers.
      ). The figures were generated using PyMOL (The PyMOL Molecular Graphics System, Version 2.4.0 Schrödinger, LLC.).

      Bio-layer interferometry (BLI) analysis

      Dissociation constant (KD), association rate constant (Kon), and dissociation rate constant (Koff) were measured on a BLItzTM system (ForteBio). 1 μM Strep-tag II-tagged NanoHA or Mini-HA/B (#1) were immobilized on OctetTM AMC Biosensors (SARTORIUS) coated with 100 μg/mL Anti-Strep-tag II tag monoclonal antibody (StrepMAB-Immuno, IBA GmbH). 30–5,000 nM of mouse E-cadherin EC1-5 purified as described previously (
      • Sugawara Y.
      • Matsumura T.
      • Takegahara Y.
      • Jin Y.
      • Tsukasaki Y.
      • Takeichi M.
      • Fujinaga Y.
      Botulinum hemagglutinin disrupts the intercellular epithelial barrier by directly binding E-cadherin.
      ) were then loaded onto the biosensors equilibrated with kinetics buffer (20 mM Tris pH 7.4, 100 mM NaCl, 2 mM CaCl2, 0.1% BSA, 0.01% Tween20). The data were analyzed by global fitting to a 1:1 binding model using BLItz Pro software (ForteBio, Version 1.2).

      Cell proliferation assays

      Caco-2, HT-29, CMT-93, MDCK, and HeLa (human cervical carcinoma-derived epithelial-like cell line) cells were seeded at 1 × 104 cells/well in 96-well plates (Iwaki), and cultured for 24 hr in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher Scientific) supplemented with 20% (for Caco-2 cells) or 10% (for HT-29, CMT-93, MDCK, HeLa cells) fetal bovine serum (FBS). The cells were further cultured with 1000 protomer nM HAs or 1% Triton X-100 at 37°C for 24 hr. Cell viability was examined using the cell proliferation reagent Cell Count Reagent SF (Nacalai Tesque) following manufacturer’s protocols.

      Data availability

      All experimental data are contained within the article.

      Supporting information

      This work contains supporting information.

      Conflict of interest disclosure

      The authors declare no conflicts of interest.

      Acknowledgments

      We thank Sachiyo Akagi for providing technical assistance and members of the Fujinaga laboratory for valuable discussion. We thank Koh Fujinaga (University of California, San Francisco) for carefully reading this manuscript. We would like to thank Editage (www.editage.com) for English language editing.

      Supplementary Data

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