Application of Genomic Editing and Microfluidics to Identify Critical Glycans Facilitating Selectin Mediated Trafficking of Human Leukocytes
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The binding of selectins (P-, L- and E-selectin) to carbohydrate epitopes expressed on leukocytes is the first step in a multi-step cell adhesion cascade that controls the rate of leukocyte recruitment at sites of inflammation. The glycans that function as selectin-ligands are post-translationally synthesized by the serial action of Golgi resident enzymes called glycosyltransferases (glycoTs). Whereas much of our current knowledge regarding the role of glycoTs in constructing selectin-ligands comes from reconstituted biochemical investigations or murine models, tools to assess the impact of these enzymes on the human ligands are relatively underdeveloped. This is significant since the selectin-ligands, particularly those that bind E-selectin, vary between different leukocyte cell populations and they are also different in humans compared to mice. This dissertation presents a modest effort to design systematically study the roles of specific glycoTs in human cell systems. To this end, a three key advances were achieved. First, novel tools were developed to genetically ablate specific glycoTs in human leukocytes using the CRISPR/Cas9 based genomic editing techniques. Second, genetically engineered primary human neutrophils lacking glycoTs were created using the lentiviral delivery of shRNA. Third, novel microfluidics based ex vivo cell adhesion assay was developed which simulated physiological hemodynamic shear environment for studying cell adhesion of the human selectins. Three hypotheses were addressed. The first part presents a comparative study on the roles of three alpha (2,3) sialyltransferases (sialylTs) in human selectin ligand biosynthesis. The results reveal that among the three enzymes (ST3Gal-3, ST3Gal-4 and ST3Gal-6) deemed relevant in literature in this regard, ST3Gal-4 is the only one that facilitate the biosynthesis of all three selectin ligands. The second part presents an attempt to delineate the contribution of glycosphingolipids in E-selectin mediated rolling of human leukocytes. Results imply that, disrupting the enzyme UDP-Glc ceramide glucosyltransferase (UGCG) that initiates the biosynthesis of extended glycosphingolipids containing sialic acid and fucose, results in low leukocyte rolling on E-selectin expressing human endothelial cells. This rolling was also accompanied by uncharacteristically high velocity and frequent cellular detachments. The third part aimed at explaining the relative roles of O-glycans, N-glycans and glycosphingolipids in regulating human leukocyte adhesion to E-selectin. It transpired from our results that all these classes of glycans collaborate to confefr E-selectin mediated human leukocyte adhesion. However, only the core-1 derived O-glycans are responsible for P- and L-selectin binding. Overall, in addition to achieving key advances in devising methods studying glycoTs, and producing novel results impacting the body of knowledge in human selectin ligands, current work also creates scope for further studies to understand mechanisms behind the differences in glycoT expression and function between humans and mice.