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.

Grant Number: 1R44GM159548-01
NIH Institute/Center: NIH
Principal Investigator: George Bendzunas