Description
- Abstract:
- Cell-surface glycans underpin key physiological mechanisms. The synthesis of these glycans relies on structurally-similar monosaccharide building blocks and multiple sites of glycosidic linkage. This results in diverse complex structures on the cell surface, each with subtly distinct carbohydrate epitopes and functionality. Characteristically, cancer cells have aberrant glycophenotypes, as tumor metabolic reprogramming leads to a variety of glycan synthesis errors. One impacted group is O-linked glycans, where the Thomsen-Friedenreich (TF) antigen (Galβ1-3GalNAcα-), a mucin-associated disaccharide expressed in ~80% of cancers, has become biomarker of interest for tumor detection due to its lack of expression in healthy tissues. The specific detection of TF at molecular resolution is vital for functional tissue profiling. However, the multitude of glycan structures necessitate novel tools for their identification and therapeutic-targeting. Glycan binding proteins (GBPs) have emerged as the leading method for profiling these diverse glycophenotypes, but lack specific structural detection. To overcome this, a previous directed evolution campaign by the Imperiali lab at MIT used a novel Sso7d-based GBP (ssoGBP) scaffold to yield an anti-TF GBP (2.4.i) with nanomolar avidity for multivalent TF. However, 2.4.i suffers from GBP-wide stereoselectivity issues; it binds the TF antigen’s stereoisomer, the Lewis C (Lec) antigen (Galβ1-3GlcNAcβ-) with just slightly worse affinity. This non-stereospecific binding limits the functionality of molecular recognition. For example, when 2.4.i is used for cancer-targeted theranostics, cells expressing other 2.4.i-recognized glycophenotypes provide off-target events that confound detection and diminish therapeutic potential. This project aims to computationally characterize the differences in 2.4.i binding to TF and Lec, informing the rational design of a stereospecific variant with significantly higher avidity for TF than Lec. Computational docking of 2.4.i to TF and Lec in HADDOCK3, restrained by experimental 1H–15N Heteronuclear Single Quantum Coherence (HSQC) Nuclear Magnetic Resonance (NMR) data, yielded hundreds of binding configurations for both antigens. Each configuration was profiled in Protein-Ligand Interaction Profiler to determine interacting residues, which work primarily through hydrogen bonding and CH-𝜋 interactions. This reveals the unique binding motifs of 2.4.i to each stereoisomer, enabling the design of stereoselective variants. The residue D34 has arisen as a potential discriminating location for TF/Lec recognition, thus the D34A mutant has been experimentally generated and its binding affinity determined through microscale thermophoresis (MST). This approach improves molecular resolution of ssoGBP carbohydrate detection, with the potential for a machine learning approach to designing ssoGBPs.
Access Conditions
- Use and Reproduction
- All rights reserved
- Rights
- In Copyright
Citation
Keean Kawamoto, Joseph J. Park, Miguel Martinez, et al.,
"Computationally-Driven Rational Design of Stereoselective Sso7d-Based Glycan Binding Proteins for Improved Cancer Therapeutics"
(2026).
Summer Research Symposium.
Brown Digital Repository. Brown University Library.
https://doi.org/10.26300/93r5-hr44
Relations
Collection:
-
Summer Research Symposium
Each year, Brown University showcases the research of its undergraduates at the Summer Research Symposium. More than half of the student-researchers are UTRA recipients, while others receive funding from a variety of Brown-administered and national programs and fellowships and go …...