Photoaffinity Probes for the Identification of Sequence-Specific Glycosaminoglycan-Binding Proteins

Glycosaminoglycan (GAG)-protein interactions mediate critical physiological and pathological processes, such as neuronal plasticity, development, and viral invasion. However, mapping GAG-protein interaction networks is challenging as these interactions often require specific GAG sulfation patterns and involve transmembrane receptors or extracellular matrix-associated proteins. Here, we report the first GAG polysaccharide-based photoaffinity probes for the system-wide identification of GAG-binding proteins in living cells. A general platform for the modular, efficient assembly of various chondroitin sulfate (CS)-based photoaffinity probes was developed. Systematic evaluations led to benzophenone-containing probes that efficiently and selectively captured known CS-E-binding proteins in vitro and in cells. Importantly, the probes also enabled the identification of >50 new proteins from living neurons that interact with the neuroplasticity-relevant CS-E sulfation motif. Several candidates were independently validated and included membrane receptors important for axon guidance, innate immunity, synapse development, and synaptic plasticity. Overall, our studies provide a powerful approach for mapping GAG-protein interaction networks, revealing new potential functions for these polysaccharides and linking them to diseases such as Alzheimer's and autism.