Synthesis and Adhesion Properties of E-Cadherin Mimetic Peptides
by Silan Toy
Date of Examination:2025-02-24
Date of issue:2025-06-12
Advisor:Prof. Dr. Claudia Steinem
Referee:Prof. Dr. Claudia Steinem
Referee:Prof. Dr. Franziska Jun.-Thomas
Persistent Address:
http://dx.doi.org/10.53846/goediss-11308
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Abstract
English
Cell adhesion molecules (CAMs) are essential for various biological processes, including tissue homeostasis and wound healing. One such molecule, the calcium-dependent protein E-cadherin, is primarily expressed at adherens junctions which connect neighboring epithelial cells. The interaction between E-cadherin molecules occurs at the N-terminal region of the monomer, where the amino acid sequence histidine-alanine-valine (HAV) in the first extracellular (EC1) domain plays a key role in mediating these interactions. To understand and mimic these E-cadherin-mediated cell-cell interactions, peptides containing the HAV-unit were synthesized using solid-phase peptide synthesis (SPPS). These HAV-peptides were then incorporated into solid-supported membranes through two distinct chemical strategies. One method involved complexing 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt) (DOGS-NTA (Ni)) lipid with a polyhistidine-tagged HAV-peptide, while the other utilized in situ Michael addition to attach a terminal Cys-HAV-peptide to a maleimide lipid. Various lengths of HAV-peptides were synthesized to investigate their impact on adhesion properties. Binding of the HAV-peptides to the membrane was verified using Reflectometric Interference Spectroscopy (RIfS) and Attenuated Total Reflectance Infrared (ATR-IR) spectroscopy, which confirmed the successful HAV-peptide attachment to the membrane. The molecular interactions between HAV-peptides and E-cadherin were investigated using Quartz Crystal Microbalance (QCM). HAV-peptides were immobilized on gold surfaces via Au-thiol coupling, and their binding to Fc-IgG-E-cadherin was studied. This allowed for a comparison of the frequency shifts induced by Fc-IgG-E-cadherin and the Fc-IgG fragment as control, enabling the calculation of the contribution from specific interactions. Additionally, holographic Video Particle Tracking (hVPT) was employed to investi-gate the behavior of HAV-functionalized beads on cell surfaces. The trajectories of the beads were analyzed, revealing that the beads exhibited strongly confined motion in the presence of HAV-peptides. This confined movement of the beads indicated a specific interaction between the HAV-peptides and E-cadherin on the cell surface, supporting the hypothesis that HAV-peptides interact with E-cadherin. To further validate the specificity of these interactions, Atomic Force Microscopy (AFM)-based colloidal probe technique (CPT) was used. From force-distance curves, the work of adhesion as well as the maximum adhesion forces could be quantified. The AFM measurements showed that the HAV-peptides significantly increased both the work of adhesion, and the maximum adhesion force compared to the absence of HAV-peptides, indicating that the presence of HAV-peptides promotes stronger adhesive interactions. Due to the complexity of the cell surface, experiments were conducted using polydimethylsiloxane (PDMS) coated with Fc-IgG-E-cadherin and micropatterned Fc-IgG-E-cadherin substrates. The experiments showed a similar trend, with both maximum adhesion force and work of adhesion showing increased values in the presence of HAV-peptides. Additionally, the known lengths of the Fc-IgG-E-cadherin molecules (24 nm for the dimeric form and 37 nm for the stretched form) were used to assign peak-to-peak distances in the force curves to the E-cadherin molecules. This study confirms that HAV-peptides enhance specific E-cadherin-mediated cell adhesion, thereby supporting their role in tissue interactions.
Keywords: E-Cadherin; Solid-Phase Peptide Synthesis; Cell junctions; Artificial cell tissue; E-Cadherin Mimetic Peptides; Colloidal probe technique AFM; Membranes
