Engineered probes for sialoglycan detection

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PROJECT SUMMARY/ABSTRACT Sialoglycans can be poor antigens, meaning that it can be challenging to develop effective antibodies for their detection. Instead, sialoglycan mapping heavily relies upon mass spectrometry, which requires expertise and instrumentation available to few laboratories. One recent push in the field has been to harvest the breadth of selectivity found within naturally-occurring sialoglycan-binding proteins in order to develop glycan-detecting proteins. This strategy identified a number of sialoglycan-binding proteins, particularly those that bind with high affinity and narrow selectivity to sialyl-T antigen (sTa). However, major gaps remain in the spectrum of the sialoglycans that are recognized. Here, we focus on sialoglycans that are likely abundant on cells or that are prevalent in disease but that lack practical probes for their detection, specifically α2,3 linked and α2,6 linked sialoglycans. We propose to create probes that recognize these glycans by tailoring the specificity of existing sialoglycan binding proteins using structure-based protein engineering. In Aim 1, we engineer the bacterial Siglec-like adhesins to create a library of probes for tri- and tetra- saccharides, each of which binds one α2,3 linked sialoglycan with high affinity and narrow selectivity. In Aim 2, we apply engineering principles to the development of probes for α2,3 linked sialoglycans and focus on the α2,6 linked sialyl Tn antigen disaccharide, a biomarker for cancer. In Aim 3, we evaluate the utility of these probes in measuring glycans in human plasma, and cross-validated these by affinity capture and mass spectrometry. The ability to distinguish glycan abundance and repertoire in healthy donors versus cancer patients will be included as a part of this aim. Successful probes will be distributed for use both in lectin arrays and in low-throughput assays.