Investigation of minimum fragment requirement for mimicking n-cadherin mediated adhesion

Cadherins are transmembrane glycoproteins that are involved in maintaining integrity of cell junctions, morphogenesis, cell sorting and many more important biological processes. In my thesis, I have focused on studying the binding affinity of a class of cadherin superfamily called N-cadherins. Prior studies have demonstrated that N-cadherins can promote neuronal growth and axonal regeneration in vitro. Moreover, there is a growing interest in development of small molecules targeting N-cadherins and designing of therapeutic agents to promote cell survival and axonal regeneration as well as inhibit cadherin mediated signaling. For in vitro studies, knowing the minimum fragments of adhesive binding domain i.e. extracellular domains that can recapitulate the wild type N-cadherin binding functions can help develop designing of biomaterials and platforms to study N-cadherin interactions. Two dimensional affinity measurements reveal probability of stable bond formation during homophilic and/or heterophilic interactions between cadherins via extracellular domains. In order to determine the minimum fragment of the extracellular domain required for binding to mimic full length cadherin and wild type cadherins expressed on cell surfaces, Micropipette Aspiration Assay (MPA) was performed. Using N-cadherin as a model for classical cadherins, the binding affinity of full length N-cadherin (N-cad EC1-5) expressed on a test cell surface (here, mesenchymal stem cell (MSCs) with the first two extracellular domains (N-cad EC1-2) and the binding pocket sequence HAVDI peptide and full length N-cadherin (N-cad EC 1-5) were measured by this assay. The first chapter of my thesis, I talked about the structural differences of most studied cadherins E and N-cadherins and briefly described the importance of the flanking amino acid of binding pocket sequence HAVDI in N-cadherins. In the following chapter, I describe the experimental approach for determining the binding affinities of N-cadherin fragments and compared the 2D binding affinity results with that of the full length wild type N-cadherin expressed on MSCs. Surprisingly, the results of the study revealed that there is no significantly difference in binding affinity between N-cad EC1-2 and N-cad EC1-5 as well as HAVDI and N-cad EC1-5. The MPA studies were conducted with both wild type MSCs expressing N-cad EC1-5 as well as RBCs modified with N-cad EC1-5. However, there is a lower binding affinity between E-cadherin and HAVDI than that between N-cadherin and HAVDI. These results demonstrate the importance of the flanking amino acids next to HAV sequence in cadherin specificity as well as the possible application of small peptide sequence HAVDI as a novel antagonist in processes involving N-cadherin mediated adhesion.

Name thesis:degree_name

M.S.

Level thesis:degree_level

Thesis

Discipline thesis:degree_discipline

Chemical Engineering

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2019

Author dc:creator

• Ahmed, Syeda Tajin

Contributors dc:contributor

• Leckband, Deborah E.

• Cadherin, binding affinity

Statement dc:rights

• © 2018 SYEDA TAJIN AHMED

Language dc:language

en

Handle dc:identifier

http://hdl.handle.net/2142/102855

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/102855