Engineered Pan-Specific Reagents for N-Glycan Detection and Enrichment

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PROJECT SUMMARY Glycans play crucial roles in nearly every aspect of biological processes, and their distinct properties make them appealing as disease biomarker targets. However, due to their highly branched and variably linked nature, glycans pose a challenge for detection, purification, and structural analysis. Although advanced analytical techniques and instrumentation have been developed that target glycans, there is still a great need for reagents with well-defined carbohydrate specificity and high affinity that can be used to independently interrogate or enrich carbohydrate samples prior to traditional analyses. Lectenz Bio has been engineering glycan-processing enzymes and glycan-binding proteins into high-affinity glycan-specific reagents with tunable properties. The Fbs1 component of the E3 ubiquitin ligase complex recognizes glycans as part of its biological role in ubiquitylation. It is therefore a promising protein for conversion into a binding reagent for asparagine-linked glycans (N-glycans), which we will achieve by employing computational modeling and directed evolution. Initial studies demonstrate that Fbs1 can be readily expressed in E. coli, and binds specifically to N-glycosylated peptides and proteins, and not to the corresponding non-glycosylated peptides and proteins. However, it does demonstrate a preference for certain N-glycan structures. A pan-specific N-glycan binding reagent would directly address the urgent need to tools to help detect glyco- biomarkers and could immediately benefit the field of glycomics/glycoproteomics by enabling sample pre- enrichment. After engineering and optimizing the production of recombinant Fbs1 and its engineered variants, Lectenz Bio will develop a suite of kits for its application in Western blotting, immunohistochemistry, ELISA, flow cytometry, and affinity chromatography.