Directed Evolution of Glycan-Binding Proteins

Abstract

Glycan-binding proteins (GBPs) are commonly used reagents for the study of glycans. They do not require specialized equipment or time-consuming experimental methods, making them widely used tools for basic research and clinical applications. Existing glycan recognition reagents,antibodies and lectins, are limited, and discovery or creation of reagents with novel specificities is time consuming and difficult.

This thesis details the generation of novel GBPs from a small, hyperthermostable DNA binding protein by directed evolution. A yeast surface display method for evolution of GBPs was developed and used to generate GBPs for the recognition of mammalian glycans sialic acid and the cancer-associated disaccharide Thomsen-Friedenreich (TF) antigen. Characterization of these proteins shows them to have specificities and affinities on par with currently available lectins. The proteins can be functionalized to create reagents that prove useful for glycoprotein blotting and cell staining applications.

Carbohydrate-protein interactions are often low affinity. Naturally occurring GBPs often oligomerize to make multivalent interaction with glycan ligands, increasing the avidity of the interaction. Fusion of the evolved GBPs to the coiled-coil trimerization domain of the lectin surfactant protein D (SP-D) leads to the formation of a trimeric GBP. These trimers are properly folded, stable, and have increased binding affinity compared to monomeric GBPs. Generation of trimeric Sso7d-based GBPs is a strategy for increasing the functional affinity of the evolved proteins, thereby making the proteins useful for a wider range of applications.

The overall goal is to create GBPs for glycans with no existing GBPs for their study. One area that can benefit from more GBP reagents is bacterial glycobiology. Many pathogens have glycans involved in virulence. One such organism is Campylobacter jejuni. The N-linked protein glycosylation pathway in Campylobacter jejuni is needed for pathogenicity of the organism, as loss of glycosylation decreases adhesion and invasion to intestinal epithelial cells and differentially modulates inflammatory responses in a gut-immune co-culture model. Future application of the developed GBP evolution platform toward bacterial glycans will have great impact on the field of bacterial glycobiology through powerful tools for studying the interactions of human pathogens, commensals and symbionts and their hosts together with novel diagnostic and analytical reagents.