PREPARATION AND CHARACTERIZATION OF GLYCAN MICROARRAYS USING SURFACE FUNCTIONALIZED CATANIONIC SURFACTANT VESICLES
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Glycan microarray technology is a promising technology to screen numerous glycan-lectin interactions in a high through put manner. Currently the focus in this topic is to create microarrays that mimic a eukaryotic cell surface, in order to simulate the glycan-lectin binding in cell-cell recognition events. Previous research in our group has been focused on characterization of catanionic surfactant vesicles with surfactants sodium dodecylbenzenesulfonate (SDBS) and cetyltrimethylammonium tosylate (CTAT). Surface functionalization of surfactant vesicles was achieved by incorporating glycoconjugates in vesicle bilayers. This thesis describes the preparation, characterization, and binding studies of glycan microarrays using functionalized catanionic surfactant vesicles.
Chapter 1 summarizes the state of glycan microarray technology and introduces the proposed strengths and limitations of the surfactant vesicle technology. Chapter 2 describes the preparation and characterization of array surfaces using Hydrophobically Modified (HM) chitosan, by electrodepositing HM chitosan on patterned gold electrodes. Surface functionalized catanionic surfactant vesicles bind selectively to HM chitosan surfaces. This chapter also describes complete characterization of the binding assay to surface deposited functionalized catanionic vesicle surfaces. Lectin binding studies conducted to monitor glucose - Con A (Conacanavalin A) binding and lactose - PNA (Peanut agglutinin) binding indicate that glycans displayed binding with their respective lectin partners. Vesicles functionalized with a complex glycan, LOS F62DA showed significant binding with antibody anti-GC mAb 2-1-L8 specific for LOS F62DA. Vesicles prepared by extracting bacterial cell membrane components can be thought of as "artificial pathogens" since they contain many of the cell surface receptors found in the pathogen itself. The binding of these `extract vesicles' with both monoclonal and polyclonal antibodies, on HM chitosan surfaces demonstrated that functionalized vesicles a model for "artificial pathogens".
Lectin binding to cell surface glycans is known to be a multidentate process. Due to this, the density of the glycan on the surface plays a key role in binding affinity and selectivity. Similarly, Our surface phase studies illustrate the effect of glycan concentration on the surface on the extent of binding on GBPs.
Next we aimed to perform enzymatic transformations on glycans incorporated in vesicles. Reaction of glycosyltransferase LgtE and UDP galactose with vesicles functionalized with LOS F62DE as well as C12 glucose functionalized vesicles. This was illustrated with up to 10 fold increase in PNA binding and ~ 50% decrease in Con A binding. LgtE shows similar reaction with LOS F62DE vesicles deposited on HM chitosan surfaces. This effect was demonstrated with the help of an ELISA assay using LOS specific antibodies. Our experiments also illustrate the ability of enzyme Endo A to transfer a tetrasaccharide Man3GlcNAc on glucose functionalized vesicles with up to 12 % efficiency in a single step. Finally, chapter 4 illustrates our preliminary efforts of creating glycan arrays by depositing surfactant vesicles on nitrocellulose surfaces.