Innovative glycan-specific reagents to accelerate the detection of disease biomarkers

PROJECT SUMMARY Glycans have several distinct properties that make them excellent targets for disease biomarkers. Firstly, because the structures of cell surface glycans are determined by a complex enzymatic pathway within the cell, any alteration to this homeostasis, such as from disease, may result in aberrant protein glycosylation. Thus, specific glycan structures that are not present, or are in low amounts, in normal states proliferate in disease states, such as cancer. Secondly, their location on cell surfaces makes them readily accessible to detection reagents. Thirdly, any change in cellular glycosylation machinery may impact a large number of glycoproteins, offering many potential glycan-related biomarkers per diseased cell. To effectively employ and discover glycan disease markers a wider range of highly-specific reagents are urgently needed. The monosaccharide mannose has been identified in many disease markers, but is difficult to detect specifically within the context of other glycans with existing reagents. Using structurally-guided mutagenesis, we will convert an α-mannosidase enzyme into a high affinity reagent for the detection of high mannose glycans that are known biomarkers for a number of cancers. Such engineered lectin-like reagents derived from enzymes are called “Lectenz®”, and have several advantages over lectins and antibodies. The principal advantages of an engineered Lectenz® over an antibody are that the Lectenz® is specific to the carbohydrate sequence, but, in contrast to antibodies, will recognize that sequence in a broad range of glycans. Further, in contrast to carbohydrate reagents based on plant lectins, engineered Lectenz® are derived from enzymes that have exquisite substrate specificities and low toxicities. Additional advantages of Lectenz® include precise definition of specificity, tunable binding properties, and ease of recombinant expression, enabling their potential use in affinity purification, western blotting, in situ histological staining, and in vivo imaging. We will generate mannose-binding Lectenz® that can bind high mannose glycans with high specificity. Glycosylation detection is essential in fully characterizing and exploiting glycans as markers of specific disease states, and yet current reagents have broad specificity.