Direct visualization of the extracellular binding structure of E-cadherins in liquid

www.nature.com/scientificreports OPEN Direct visualization of the extracellular binding structure of E‑cadherins in liquid Teiko Shibata‑Seki1, Masato Nagaoka1, Mitsuaki Goto1*, Eiry Kobatake2 & Toshihiro Akaike1 E-cadherin is a key Ca-dependent cell adhesion molecule, which is expressed on many cell surfaces and involved in cell morphogenesis, embryonic development, EMT, etc. The fusion protein E-cad-Fc consists of the extracellular domain of E-cadherin and the IgG Fc domain. On plates coated with this chimeric protein, ES/iPS cells are cultivated particularly well and induced to differentiate. The cells adhere to the plate via E-cad-Fc in the presence of Ca2+ and detach by a chelating agent. For the purpose of clarifying the structures of E-cad-Fc in the presence and absence of Ca2+, we analyzed the molecular structure of E-cad-Fc by AFM in liquid. Our AFM observations revealed a rod-like structure of the entire extracellular domain of E-cad-Fc in the presence of Ca2+ as well as trans-binding of E-cad-Fc with adjacent molecules, which may be the first, direct confirmation of trans-dimerization of E-cadherin. The observed structures were in good agreement with an X-ray crystallographic model. Furthermore, we succeeded in visualizing the changes in the rod-like structure of the EC domains with and without calcium. The biomatrix surface plays an important role in cell culture, so the analysis of its structure and function may help promote cell engineering based on cell recognition. Cadherin is a group of cell surface glycoproteins, identified and named as a calcium-dependent cell adhesion molecule1. There are two types of cell adhesion, cell–cell adhesion and cell-substrate adhesion. Cadherin forms the former type of homophilic adhesion. There are more than 100 types of cadherins, among which classical cadherins are the most studied. Classical cadherins are single-pass transmembrane proteins of the cell membrane. On the cytoplasmic side, cadherins link to actin cytoskeleton through such molecules as catenin groups and EPLIN. Many physiological studies have been carried out to show that cell–cell adhesion is controlled from the cytoplasmic side2–6. The extracellular site is composed of five domains bound in tandem (Fig. 1a), and the size of one domain is about 110 amino acid residues, which are named Extra Cellular 1 (EC1) -EC5 in order from the N-terminal side. Cadherins bind by binding calcium ions to calcium ion binding sites between individual extracellular domains. The EC1 and EC2 domains are the ones directly involved in the cell adhesion, and there have been many studies on the interactions between them. E-cadherin-Fc (E-cad-Fc) is a fusion protein, which consists of the extracellular domain of E-cadherin and the IgG Fc d omain7–10 (Fig. 1e). E-cadherin involves in cell morphogenesis, embryonic development, cancer, EMT, etc.11–15 so that the chimeric proteins may serve as important biomaterial which helps clarify the cell function. On plates coated with E-cad-Fc, ES/iPS cells are cultivated particularly well and induced to differentiate with several methods16–19. Application of E-cad-Fc to particle coating is also in progress20. Chimeric proteins of the same kind, namely N-cad-Fc and VE-cad-Fc, have been also developed, which also promote differentiation of neural progenitor c ells21–24 and human umbilical vein endothelial cells (HUVECs)25 respectively in vitro. Experiments in mice aimed at developing treatments for newborn brain injury showed that N-cad-Fc-coated gelatin sponge artificial scaffolds promote neuronal regeneration and functional recovery, highlighting the significance of biomaterials also in vivo26. Cells cultivated on plates coated with these cadherin chimeric proteins adhere in the presence of Ca2+ and detach when Ca2+ is removed by a chelating agent such as EDTA and EGTA. It is of great importance to clarify structures of the chimeric proteins in the presence and absence of calcium ions, as well as their cell adhesion mechanism, so that the chimeric proteins may be better used for biomaterial development. With these backgrounds, we focus on morphological observations of E-cad-Fc by using AFM (Atomic Force Microscope). AFM allows direct observation of a sample as it is. Unlike an electron microscope, samples do not need to be dried or frozen, and can be measured in a physiological environment. Furthermore, there is no 1 Biomaterials Center for Regenerative Medical Engineering, Foundation for Advancement of International Science, 24‑16 Kasuga, 3‑chome, Tsukuba, Ibaraki 305‑0821, Japan. 2School of Life Science and Technology, Tokyo Institute of Technology, G1‑13, 4259, Nagatsuta, Midori‑ku, Yokohama, Kanagawa 226‑8502, Japan. *email: goto@ fais.or.jp Scientific Reports | (2020) 10:17044 | https://doi.org/10.1038/s41598-020-72517-2 1 Vol.:(0123456789) www.nature.com/scientificreports/ Figure 1.  E-cadherin model. (a) Schematic illustration of E-cadherin binding. The extracellular site of E-cadherin is composed of five domains bound in tandem. The binding model is based on crystal structure (PDB ID: 3Q2V28). (b) Crystallography structural model (PDB ID: 3Q2V28) of E-cadherin extracellular domains created with PyMOL. (c) Crystallography structural model (PDB ID: 5JII44) of IgG-Fc domain created with PyMOL. (d) E-cadherin-Fc molecular model created by combining two crystallography structural model (b,c). (e) Schematic illustration of E-cadherin-Fc molecular model. EC5 domain is connected to Fc fragment. Calcium ions are shown as blue spheres. need for sample staining. With AFM observation, a resolution of nm level is expected. For example, the periodic double-helix structure of B-DNA in the solution was observed at a spatial resolution of 1.2 nm with the PeakForce Tapping mode27. The short axis diameter of the EC domain is expected to be around 2 nm (calculated from PDB ID: 3Q2V28, Fig. 3b), so that AFM may be well suited for our study to capture E-cad-Fc structure in cell culture. Since cadherins are membrane protein and tend to aggregate and are difficult to analyze as a whole molecule, previous analyses were limited to their hydrophilic EC domains. To the best of authors’ knowledge, the first AFM observation of cadherins in liquid was performed by Baumgartner et al.29 on VE cadherin which belongs to the type II classical cadherin family. Later, Brasch et al.30. obtained AFM images of molecules under ambient condition which are presumed to be monomers and dimers of VE cadherin. Harrison et al.31 made an AFM observation in liquid for the entire extracellular domain of N-cadherin which belongs to the type I classical cadherin family. For our observations, E-cad-Fc is employed, which is soluble and easy to handle. E-cad-Fc molecules in solution may take two states: in one state, two EC domains within the same E-cad-Fc molecule interact laterally, while the EC domain trans-interacts with different E-cad-Fc molecules in the other state. In other (...truncated)