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Abstract
Cell surface glycans on glycoproteins and glycolipids modulate cellular interactions in development and disease. Interactions between specific glycans and their cognate glycan binding proteins enhance or suppress inflammatory responses in many vertebrate tissues. Many of these interactions occur at epithelial surfaces, such as the epithelial lining of the airway and the digestive tract. This thesis describes enhanced methods to analyse subsets of glycoprotein glycans that frequently participate in inflammatory responses and applies these methods to the characterization of normal human airway epithelial cells grown and differentiated at an air-liquid interface. This thesis also describes the first comprehensive glycomic characterization of an aggressive mouse model of Alzheimers disease (AD) and identifies a previously uncharacterized change in the expression of anti-inflammatory glycans at the epithelial interface between the vascular system and the cerebrospinal fluid (CSF). To assess the glycomic changes associated with amyloidopathy of AD, we characterized protein and lipid glycosylation in the highly aggressive 5xFAD mouse model and detected broad changes in glycoprotein and glycolipid glycosylation by multi-dimensional mass spectrometry. These changes were verified and extended orthogonally using structurally specific plant lectin probes on blots and in tissue sections. We also detected changes in the expression of glycan structures recognized by SIGLEC-F, an anti-inflammatory glycan binding protein expressed on mouse macrophages, eosinophils. microglia, and subsets of T-cells. Engagement of SIGLEC-F by its glycan ligands induces apoptosis in eosinophilic asthma models and supports anti-inflammatory polarization of macrophages. Significantly increased expression of SIGLEC-F glycan ligands was detected in the 5xFAD mouse at the apical surface of the epithelial cells of the choroid plexus (ChP), the ventricular structure responsible for forming CSF and for modulating immune cell trafficking into the brain (Meeker et al. 2012; Lehtinen et al. 2013). Glycan-based immunoprecipitation to identify brain glycoproteins that carry glycans recognized by SIGLEC-F identified two candidate SIGLEC-F counter-receptor proteins, Lipoprotein Receptor Related Protein 1 (LRP1) and Versican (VC). While LRP-1 is significantly increased at the interface between the basal surface of ChP epithelial and endothelial cells in the 5xFAD mouse, it does not colocalize with the distribution of SIGLEC-F ligands. Nonetheless, glycoform differences were detected in comparing the PNGaseF sensitivity and antibody epitope masking of LRP1 in 5xFAD mouse brain compared to control. Like LRP1, VC is also strongly expressed at the endothelial face of the ChP but does not colocalize with SIGLEC-F ligands. Thus, these two proteins likely function as major SIGLEC-F counter-receptors in whole brain, but the specific counter-receptor at the ChP epithelium remains to be determined. Significant increases in the extravasation of bone marrow derived M2 macrophages (anti-inflammatory macrophages) were detected in the 5xFAD mouse ChP, indicating that the glycomic changes at this essential epithelial interface may mediate recruitment or differentiation of specific inflammatory cells.