Exploiting nature's glycan-transfer enzymes to investigate glycan function

Abstract

Glycans are an important class of biological macromolecules that have diverse roles within the cell and on the cell surface. They are involved in immunological functions, cancer biology and stem cell biology to name a few examples. Due to their dynamic nature in response to their environment, glycans can be considered a biomarker for the overall fitness of the cell. The ability to image and characterize these dynamic changes to the glycome would advance our understanding of the detailed roles of glycans in biological processes.;Standard methods for glycan detection suffer from low specificity (i.e. antibodies and lectins), or are limited to the study of monosaccharides (i.e. metabolic oligosaccharide engineering). It is highly desirable to create specific labeling methods to probe the functions of higher order glycans because they encode information for specific cell-surface molecular recognition.;Recently, a highly specific chemoenzymatic approach for the tagging of N-acetyl lactosamine (LacNAc) with GDP-fucose analogs to generate a chemically tagged Lewis X was developed using an alpha1,3fucosyltransferase enzyme from H. plyori. Lewis X is a major binding partner for the DC-specific intracellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN) on the surface of human dendritic cells. In the first part of this thesis, a magnetic, glyco-nanoparticle decorated with Lewis X was generated and utilized to enrich for dendritic cells bearing DC-SIGN in a heterogeneous cell population.;In an effort to visualize the specific location of LacNAc, the chemoenzymatic method was then translated to a histology method dubbed CHoMP---Chemoenzymatic Histology of Membrane Polysaccharides. CHoMP analysis of LacNAc on human lung adenocarcinoma samples revealed a sharp decrease in LacNAc expression levels between normal and grade one patient samples, suggesting a potential application of this technique in early cancer diagnosis.;CHoMP analysis of mouse intestines showed a strong labeling pattern on the Paneth cells, cells responsible for maintaining the stem cells niche. In the final chapter of this thesis, glycosyltransferases were applied towards generating a sugar "blocking" system in crypt organoid cultures. "Blocking" of LacNAc resulted in increased proliferation of the organoid cultures, likely due to the absence of binding by galectin-2, a negative regulator of proliferation.