UTILIZATION OF FUNCTIONALIZED CATANIONIC SURFACTANT VESICLES TO INVESTIGATE PROTEIN-GLYCAN INTERACTIONS
Mahle, Amanda Caroline
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Despite the estimate that more than half of the human proteome is glycosylated, the field of glycomics lags substantially behind proteomics and genomics. The ubiquity of carbohydrates in and on the cell is directly related to the extensive roles they play in the folding and stabilization of protein structure, and in cellular recognition processes. The slow progress in deciphering glycan interactions at a molecular level is in large part due to the absence of a functional system to express, on a large scale, carbohydrates of known structure, in the context of a biologically relevant assay system. The goals of my research are to prepare and characterize glycan-functionalized catanionic surfactant vesicles as a platform for glycan synthesis, and to demonstrate that the resulting glycan-functionalized vesicles serve as a scaffold for the interrogation of protein-glycan interactions. To accomplish this goal, a high-yield method for the purification of N. gonorrhoeae lipooligosaccharide (LOS) glycosyltransferase LgtE, an enzyme that catalyzes the addition of galactose onto a terminal glucose found on LOS, was first optimized. Preliminary evidence is presented, suggesting that catanionic vesicles solubilize macromolecules found in inclusion bodies, indicating that surfactant vesicles facilitate the purification of insoluble species. A novel method exploiting differential lectin binding, measured by flow cytometry, was developed to demonstrate LgtE activity on whole cell bacteria and on catanionic vesicles functionalized with LOS or a synthetic glycolipid acceptor substrate. The data from these studies confirm the vesicles' robustness in the highly sensitive assay, and demonstrates LgtE mediated oligosaccharide biosynthesis on vesicles, regardless of acceptor origin. Enzyme activity was then characterized on whole cells and LOS functionalized vesicles. Unexpectedly, LgtE is observed to have approximately the same affinity for both terminal glucose and galactose as acceptor substrates for galactosyl transfer. Finally, enzymatic synthesis and retention of the vesicles by hydrophobically modified chitosan coated electrodes is demonstrated by differential antibody binding. This dissertation presents proof-of-concept that glycan-functionalized catanionic vesicles can be used to create a high-specificity and high-throughput glycan array. This array will allow for the investigation of a variety of protein-glycan interactions, and will undoubtedly have applications in many fields of glycomics.