Cells can mediate intercellular communication by the secretion and uptake of microvesicles, nano-sized membranous particles that carry signaling molecules, antigens, lipids, mRNA and miRNA between cells. The biological function of these vesicles is dependent upon their composition and cellular origin which is regulated by mechanisms that are not well understood. Based on their molecular content, microvesicles may play a role in immune regulation, cancer progression, the spread of infectious agents and numerous other important normal and pathogenic processes. The proteomic content of microvesicles from diverse sources has been intensely studied. In contrast, little is known about their glycomic content. The glycosylation pattern of a protein or lipid plays a key role in determining its functional properties in several ways. Glycans can determine the trafficking of a protein to particular regions of the cell as well as the protein’s half life. In addition, the glycan-dervied oligomerization of glycolipids and glycoproteins is a known mechanism for the activation of receptors and recognition of ligands on the surface of the cell. Glycomic analysis may thus provide valuable insights into microvesicle function. I utilized lectin microarray technology to compare the glycosylation patterns of microvesicles derived from a variety of biological sources. When compared to cellular membranes, microvesicles were enriched in high mannose, polylactosamine, α2-6 sialic acid, and complex N-linked glycans but exclude terminal blood group A and B antigens. The polylactosamine signature in microvesicles from different cell lines derives from distinct glycoprotein cohorts. After treatment of Sk-Mel-5 cells with lactose to inhibit lectin-glycan interactions, secretion of microvesicle resident proteins was severely reduced. Taken together, this work provides evidence for a role of glycosylation in microvesicle-directed protein sorting. / text

The cell surface is enveloped with a myriad of carbohydrates that form complex matrices of oligosaccharides. Carbohydrate recognition plays crucial and varying roles in cellular trafficking, differentiation, and bacterial pathogenesis. Lectin microarray technology presents a unique platform for the high-throughput analysis of these structurally diverse classes of biopolymers. One significant hinderance of this technology has been the limitation imposed by the set of commercially available plant lectins used in the array. To enhance the reproducibility and scope of the lectin panel, our lab generated a small set of bacteria-derived recombinant lectins. This dissertation describes the unique advantages that recombinant lectins have over traditional plant-derived lectins. The recombinant lectins are expressed with a common fusion tag, glutathione-S-transferase (GST), which can be used as an immobilization handle on glutathione (GSH)-modified substrates. Although protein immobilization via fusion tags in a microarray format is not novel, our work demonstrates that protein activity through site-specific immobilization is enhanced when the protein is properly oriented. Although orientation enhanced the activity of our GST-tagged recombinant lectins, the GSH-surface modification precluded the printing of non-GST-tagged lectins, such as the traditional plant lectins, thus limiting the structural resolution of our arrays. To solve this issue, we developed a novel print technique which allows the one-step deposition and orientation of GST-tagged proteins in a microarray format. To expand our view of the glycome, we further adapt this method for the in situ orientation of unmodified IgG and IgM antibodies using GST-tagged antibody-binding proteins. Another advantage of recombinant lectins is in the ease of genomic manipulation, wherein we could tailor the binding domain to bind a different antigen. We demonstrate this by producing non-binding variants of the recombinant lectins to act as negative controls in our microarrays. Along with the non-binding variants, we developed a lectin displayed on the surface of phage. In the hopes generating more novel lectins, I will describe our current efforts of lectin evolution using phage-displayed GafD. By generating novel tools in lectin microarray technology, we enhance our understanding of the role of carbohydrates on a global scale. / text