Investigating the interaction between the tetraspanin CD82 and gangliosides, and, The binding partners of CLEC14A and the function of the CLEC14A extracellular domain

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Lodhia, Puja (2012). Investigating the interaction between the tetraspanin CD82 and gangliosides, and, The binding partners of CLEC14A and the function of the CLEC14A extracellular domain. University of Birmingham. M.Res.

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Abstract

Project 1: CD82 is a tetraspanin involved in tumour metastasis suppression. It is downregulated in several cancers including breast, liver and prostate cancer. Although interactions with gangliosides and cholesterol have been demonstrated, no ligand has yet been identified to directly target CD82. CD82 interaction with gangliosides has been explored by immunoprecipitation from cell lysates. However, direct interaction of purified full length CD82 protein with ganglioside has not yet been shown. This study demonstrates the interaction of full length CD82 protein with the ganglioside GM2 by ELISA, and did not require the presence of any other components of the cell membrane. This interaction was further explored by surface plasmon resonance and solid state NMR techniques.

Project 2: CLEC14A is tumour endothelial marker (TEM). It is upregulated by reduced shear stress and facilitates angiogenesis. However, the biological activity of CLEC14A is poorly characterised. This study validates two binding partners of CLEC14A by co-immunoprecipitation and mass spectrometry. The activity of the CLEC14A extracellular domain (ECD) was also investigated. Flow cytometry revealed that CLEC14A-ECD is able to bind to the surface of human umbilical vein endothelial cells (HUVECS). However, treatment of HUVECS with 20 ug/ml CLEC14A-ECD did not affect cell motility in scratch wound assays. The cleavage site of CLEC14A by the intra-membrane serine protease rhomboid-like protein 2 (Rhbdl2) was also explored. These results will increase the understanding of the biological activity of CLEC14A and will aid the intelligent design of drugs targeting CLEC14A to inhibit tumour angiogenesis.

Type of Work:

Thesis (Masters by Research > M.Res.)

Award Type:

Masters by Research > M.Res.

Supervisor(s):