Binding studies of E-Cadherin peptides to the EC1 domain of E-Cadherin

Abstract

The blood brain barrier (BBB) is a key role in delivering medication to the brain; if the drug molecules can overcome this obstacle, many brain diseases (i.e., Alzheimer's, Parkinson's, and brain tumor) can be effectively treated. E-cadherin is important in forming cell-cell adhesion in intercellular junctions of the biological barriers (i.e., intestinal mucosa and BBB). Cadherin peptides (HAV6 and ADTC5) have been shown to modulate the BBB in the cell culture and in in-situ rat brain perfusion models. The hypothesis is that cadherin peptides modulate the tight junctions of the BBB by binding to the EC1 domain of E-cadherin. Thus, the objective of this project is to determine the dissociation constants (Kds) of linear and cyclic cadherin peptides (i.e., HAV- and ADT-peptides) to the expressed EC1 domain of E-cadherin using circular dichroism (CD) spectroscopy. The data show that the cyclic HAVc3 peptide (Kd = 66.7±18.0 nM) has better binding property to the EC1 domain than the linear HAV6 peptide (Kd = 120.1±11.9 nM). Cyclic ADTC5 (Kd = 50.2± 11 nM) has lower dissociation constant than ADTC1 (Kd = 119.7± 16 nM). Mutation of the valine residue in ADTC5 to glutamic acid (ADTC7; Kd = 0.43± 0.050 nM) and threonine (ADTC8; Kd = 0.45±0.012) makes the peptides to have 100-fold tighter binding compared to the EC1 domain. Mutation of the valine residue in ADTC5 to a tyrosine residue (ADTC9; Kd = 0.038± 0.009) increases binding to the EC1 domain by 1000-fold. Finally, mutation of the valine residue to arginine demolishes the activity of ADTC10. In the future, the activity of ADTC9 to modulate the BBB will be compared to ADTC5 in in vitro and in vivo models of the BBB.