Semaphorin 3A binding to chondroitin sulfate E enhances the biological activity of the protein, and cross-links and rigidifies glycosaminoglycan matrices

Semaphorin 3A (Sema3A) is a secreted protein that signals to cells through binding to neuropilin and plexin receptors and provides neurons with guidance cues key for axon pathfinding, and also controls cell migration in several other biological systems. Sema3A interacts with glycosaminoglycans (GAGs), an interaction that could localize the protein within tissues and involves the C-terminal domain of the protein. This domain comprises several furin cleavage sites that are processed during secretion and in previous works have hampered recombinant production of full-length wild type Sema3A, and the biochemical analysis of Sema3A interaction with GAGs. In this work, we have developed a strategy to purify the full-length protein in high yield and identified two sequences in the C-terminal domain, KRDRKQRRQR and KKGRNRR, which confer to the protein sub nM affinity for chondroitin sulfate and heparan sulfate polysaccharides. Using chemically defined oligosaccharides and solid phase binding assays, we report that Sema3A recognizes a (GlcA-GalNAc4S6S)2 motif but not a (GlcA2S-GalNAc6S)2 motif and is thus highly specific for type E chondroitin sulfate. Functionally, we found that Sema3A rigidified CS-E films that mimic the GAG presentation within extracellular matrices (ECMs), suggesting that Sema3A may have a previously unidentified function to cross-link and thus stabilize GAG-rich ECMs. Finally, we demonstrated that the full-length Sema3A is more potent at inhibiting neurite outgrowth than the truncated or mutant forms that were previously purified and that the GAG binding sites are required to achieve full activity. The results suggest that Sema3A can rigidify and cross-link GAG matrices, implicating Sema3A could function as an extracellular matrix organizer in addition to binding to and signaling through its cognate cell surface receptors.