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

Abstract

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 polysaccharidebased 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 benzophenonecontaining 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.

Deciphering Structural Determinants in Chondroitin Sulfate Binding to FGF-2: Paving the Way to Enhanced Predictability of Their Biological Functions

“…On the other hand, given the large number of binding partners and diverse GAG structures, new approaches need to be developed that enable us to look more rapidly and more globally across protein families and GAG classes [ 14 ]. Very recently, we have proposed a new “holistic approach” to look globally at how particular sulfate distribution within the disaccharide epitope directs the binding of chondroitin sulfates to growth factors.…”

Photoaffinity Labeling Chemistries Used to Map Biomolecular Interactions

“…For example, a benzophenone‐based probe of chondroitin sulfate enriched 54 proteins from cortical neurons and helped identify common chondroitin sulfate recognizing protein motifs (Figure 9B). [18] In this case, the photoactive glycopolymer was accessed by chemical synthesis, which affords flexibility for the incorporated PAL chemistry. Several aryl trifluoromethyl diazirine and benzophenone probes were prepared, and the benzophenone probes were selected for further use due to their higher labeling yields.…”

On the Selectivity of Heparan Sulfate Recognition by SARS-CoV-2 Spike Glycoprotein

“…Various other challenges regarding HS oligosaccharides will have to be overcome including synthesis and off-target effects. This includes elucidating the interactome of the sequences identified in this study, which is difficult because the GAG interactome technology is still being developed . In the interim, screening against proteins known to bind GAGs against 31 hexasaccharides of Table S2 would be advisible.…”