N-Sulfated Heparan Sulfate Promotes Reelin Signaling as a Co-receptor

Heparan sulfate (HS) plays a central role in signal transduction, while Reelin is an essential signaling protein in both the developing and adult brain. A Reelin COLBOS variant was recently discovered with enhanced HS binding and resilience against autosomal dominant Alzheimer's disease (ADAD), underscoring the importance of Reelin-HS interactions. However, the glycan determinants of Reelin-HS interactions have not been well-characterized, which we systematically investigated here. Surface plasmon resonance (SPR) showed that full length Reelin binds HS with high affinity (<i>K</i>_D = 17 ± 5 nM), which is enhanced by the COLBOS variant (<i>K</i>_D = 10 ± 2 nM). Competition SPR and glycan array studies further revealed that HS N-sulfation is critical for Reelin-HS binding, consistent with Haddock modeling. In cell surface binding assays, heparinase treatment, which degrades HS, or the knockout of a key HS N-sulfation enzyme (NDST1) significantly reduced Reelin attachment. Functionally, a cellular split-luciferase assay showed that heparinase treatment or adding heparin in culture medium reduces Reelin-induced ApoER2 dimerization, demonstrating that HS is a coreceptor for Reelin receptor activation. In contrast, N-desulfated heparin does not inhibit Reelin receptor dimerization. Our work establishes HS as a coreceptor for Reelin signaling and N-sulfation as a key glycan determinant of Reelin-HS recognition. Our work provides mechanistic insights into diverse neurodevelopmental and neurodegenerative diseases associated with Reelin signaling and suggests novel therapeutic strategies targeting HS sulfation.