Customizable 3D Matrix to Investigate Glycan Function

Customizable 3D Matrix to Investigate Glycan Function PI: Matthew Brichacek; Co-I: William Gramlich, University of Maine Abstract/Summary Glycans in the form of glycoproteins, proteoglycans, and glycolipids provide structural support, mediate intrinsic interactions, and facilitate extrinsic recognition allowing glycans play an integral role in cellular

pathways and disease states. Interactions of the glycans with glycan binding proteins enable these important and diverse biological functions. However, the multivalent nature of glycan-protein interactions, as well as the dependence on distance and orientation, have prevented the Glycoscience community from realizing the full

therapeutic potential of glycans. The proposed, customizable glycan-modified material will enable the identification and characterization of glycans relevant to human health and explore their effects in a controlled and customizable extracellular environment. The first component of the customizable 3D matrix is a suite of thiol-functionalized glycans. Homogeneous glycan

substrates can be tagged with a thiol functionality using reductive amination at the reducing-end of commercially available glycans or those obtained after fractionation of natural samples. Heterogeneous glycan pools can be similarly prepared after oxidative or enzymatic cleavage of saccharides from biological samples. The thiol-

functionalized glycans produced offer distinct advantages over existing probes in the efficiency and spatiotemporal control of the conjugation techniques available. These thiol-modified glycans are first validated using a microarray of the thiol-functionalized glycans attached to a glass surface. Glycan microarrays are a

sensitive and high-throughput tool to evaluate protein-glycan binding by screening glycan binding proteins, antibodies, whole cells, or viruses. Previous microarray investigations have identified and quantified glycan- protein interactions using direct or indirect detection of fluorescence. However, the proposed research plan

utilizes a customizable hydrogel matrix that enables the glycan-protein interaction to be meticulously characterized: 1) three-dimensional environment effects 2) concentration dependent behavior; and 3) synergistic or antagonistic interactions with other glycans. The proposed diagnostic material system is a new tool to better understand the roles that glycans play in human

physiology and disease. The identification and characterization of glycan binding proteins will validate new drug targets and diagnostic markers in disease states and have a profound effect on human health.