Characterising the molecular interaction between CLEC14A and Multimerin 2

Baber, Aleen (2024). Characterising the molecular interaction between CLEC14A and Multimerin 2. University of Birmingham. Ph.D.

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Abstract

The C-type lectin domain group 14A (CLEC14A) protein, a single pass transmembrane spanning glycoprotein, is an endothelial expressed protein found to be highly and selectively expressed on tumour vessels. CLEC14A interacts with various ligands including the extracellular matrix protein Multimerin 2 (MMRN2) and heparan sulfate polysaccharides. CLEC14A’s interaction with MMRN2 elicits a proangiogenic phenotype and upon blocking this interaction there is a reduction in tube formation and cell migration in vitro and tumour size in vivo. The aim of this project was to further characterise the interactions of CLEC14A with MMRN2 and heparin as well as explore the structure of CLEC14A and MMRN2. To this end an alanine-scanning mutagenesis approach, coupled with AlphaFold predictive modelling, determined that residues S137, T139 and R141 of human CLEC14A directly interact with MMRN2. The R161 residue of CLEC14A was also shown to partially contribute to the interaction with heparin. It was revealed that the R100 and R141 residues of CLEC14A form part of the binding epitope for the CRT4 antibody which blocks the interaction between CLEC14A and MMRN2. The CRT4 antibody, known to modulate tumour vessel formation, also blocked the interaction between CLEC14A and heparin, thus expanding our understanding of how this reagent is acting to mediate its biological effects. Additionally, analytical ultracentrifugation revealed that MMRN2 forms a trimer in solution, providing new insights into how CLEC14A may bind to MMRN2. Taken together, the data presented in this thesis expands on the current understanding of the interaction between CLEC14A and MMRN2 and heparan sulfate, providing valuable strategies that could be used to better determine how different ligands affect the biology of CLEC14A. This may ultimately give rise to novel therapeutic strategies to target CLEC14A in cancer.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):