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 ?2,3 linked and ?2,6 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 ?2,3 linked sialoglycan with high affinity and narrow selectivity.
In Aim 2, we apply engineering principles to the development of probes for ?2,3 linked sialoglycans and focus on the ?2,6 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.
Probes that detect specific glycan structures on cells can enhance our understanding of processes mediated by glycosylation or can be used as diagnostic tools in disease states with altered glycosylation. However, there are few practical reagents for the detection of sialoglycans, particularly ?2,3 and ?2,6 linked sialoglycans, both of which are biomarkers for cancer. Here, we propose to develop such probes using protein engineering.
