A generalizable mechanism of CD24 signalling and its ability to specifically alter the biogenesis of B cell extracellular vesicles

Ayre, D. Craig (2017) A generalizable mechanism of CD24 signalling and its ability to specifically alter the biogenesis of B cell extracellular vesicles. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

CD24 is a variably glycosylated, glycophosphatidylinositol (GPI)-anchored cell surface protein. Its expression is dynamic during cellular differentiation and ligand interaction. While several decades of research have established that CD24 engages in many cell-type specific functions in many cases the ligands of CD24 are unknown, and researchers have relied on the use of antibodies to mimic ligand binding. Furthermore, as a GPI-anchored protein, CD24 must rely on in cis signalling partners, however little has been elucidated on the cell membrane-proximal signalling activities of CD24. Therefore, the work presented in this thesis presents a more comprehensive examination of CD24 expression, and function in multiple cell types, followed by an in-depth analysis in immature B lymphocytes. In B cells, CD24 is known to mediate the induction of apoptosis. To predict in cis and in trans partners of CD24, an analysis of CD24 mRNA expression, and its potential ligands was performed. In some tissues, such as B cells, an association was identified between CD24 and putative ligands, including Siglec-2. In other tissues, no significant associations were identified. Our previous investigation suggested that CD24 is involved in vesicle trafficking, Consistent with this, CD24 surface protein expression was shown to be dynamic within 1 h of Ab stimulation in WEHI-231 immature B cells and in ex vivo primary immature B cells. I found CD24 promotes the generation of plasma membrane-derived microvesicles (MVs). These MVs transported CD24 between cells. MVs also carried a variety of nucleic acid cargo, identified by RNA-Seq, and protein cargo as determined by mass spectrometry and flow cytometry. The incorporation of these cargos into MVs was variably influenced by CD24 stimulation. Overall, these data suggest that MVs generated in response to CD24 play a role in regulating mitochondria, and immune cell activation. Finally, a unifying hypothesis on the function of CD24 is presented herein, proposing its role as a moderating rheostat of cellular signalling rather than a de novo signalling receptor. Together, this work has significantly advanced our understanding of CD24 in B cells, and may provide insight for studies in other cell types or in diseases such as leukemia.

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