Abstract

CD47 is a 47-52 kDa membrane spanning glycoprotein, which has 5 known isoforms and is a member of the immunoglobulin superfamily (IgSF). It possesses a large extracellular N-terminal IgV like domain. CD47 acts as a ‘do not eat me’ signal and consequently as a marker of self. Moreover, CD47 is reported to be upregulated in cancer cells allowing them to escape immune surveillance by binding to SIRP alpha on macrophages. The aims of this thesis were to evaluate and to confirm the presence of CD47 isoforms in erythroid and erythroleukemic cells, identify new CD47 binding proteins that may mediate the phosphatidylserine exposure pathway (eryptosis is the process of red cell death which involves the switch of phosphatidylserine from the inner leaf of the membrane to the outer leaf), investigate the functions of the red cell CD47 complexes identified, and to characterize seven different monoclonal antibodies (mAbs) against CD47 that could be used in anti-cancer therapy. To achieve these aims, Next Generation Sequencing (NGS), flow cytometry to measure phosphatidylserine exposure, western blot analysis, immunoprecipitation (IP) of CD47, and Mass Spectrometry (LC-MS/MS) were used. Results showed the presence of all CD47 five isoforms in erythroid and erythroleukemic cells, and their relative proportions have been assessed. The correct transcript sequence for CD47 isoform 5 was confirmed. CD47 isoform 2 was the most abundant isoform expressed in erythroid and erythroleukemic cells. Flow cytometry results showed three different levels of phosphatidylserine exposure by the seven different monoclonal antibodies which recognise epitopes on the N-terminus of CD47 (BRIC 32, BRIC 122, BRIC 124, BRIC 125, BRIC 126, BRIC 168 and BRIC 211). A novel finding was that a mAb to glycophorin A switched off the eryptotic effect of all anti-CD47 N-terminal monoclonal antibodies. Mass Spectrometry results showed novel protein:protein interactions in samples immunoprecipitated (IP) using N-terminal and C-terminal antibodies against CD47. S100 proteins were found to be up-regulated in CD47 IPs and five lipid raft components were found up-regulated in CD47 IPs when compared against normal erythrocyte membranes (’ghosts’). This study concludes that the CD47 eryptosis pathway involves an interaction with S100 proteins that leads to calcium influx and subsequent enhancement of protein kinase C action, which is involved in the eryptosis pathway. One CD47 monoclonal antibody, BRIC 32, could be a potential therapeutic anti-cancer agent as it shows a low level of eryptosis in erythrocytes. The novel switch-off eryptotic effect of anti-CD47 monoclonal antibodies caused by anti-glycophorin A (mAb) BRIC 256 might play a role in eryptosis pathway worthy further investigation. This pathway may well be another useful avenue to explore in cancer therapy.

Keywords

CD47, Eryptosis, Apoptosis, Next Generation Sequencing, Flow-Cytometry, Cancer Therapy

Document Type

Thesis

Publication Date

2019

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